UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Sex differences in the integrated response to high respiratory muscle work during exercise Dominelli, Paolo Biagio 2016

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2017_february_dominelli_paolo.pdf [ 2.59MB ]
Metadata
JSON: 24-1.0340564.json
JSON-LD: 24-1.0340564-ld.json
RDF/XML (Pretty): 24-1.0340564-rdf.xml
RDF/JSON: 24-1.0340564-rdf.json
Turtle: 24-1.0340564-turtle.txt
N-Triples: 24-1.0340564-rdf-ntriples.txt
Original Record: 24-1.0340564-source.json
Full Text
24-1.0340564-fulltext.txt
Citation
24-1.0340564.ris

Full Text

   Sex differences in the integrated response to high respiratory muscle work during exercise.  by  Paolo Biagio Dominelli B.H.K., The University of British Columbia, 2010 M.Sc., The University of British Columbia, 2012  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Kinesiology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2016  © Paolo Biagio Dominelli 2016 ii  Abstract Purpose.   The purpose of this thesis was to investigate sex differences in the integrated response to high respiratory muscle work during exercise.  To accomplish this, I developed two novel methodologies (Chapters 2 & 4) in order to answer two subsequent research questions (Chapters 3 & 5). Methods. Chapter 2:  Using computer software coupled with physiological measurements, I measured the oxygen cost of exercise hyperpnea in healthy subjects.  Chapter 3:  Healthy men and women performed voluntary hyperpnea while the oxygen cost of breathing was determined.  The absolute and relative oxygen cost of breathing was compared between the sexes at different absolute and relative ventilation. Chapter 4: Using readily available components, I developed a proportional assist ventilator that could operate during all exercise intensities. Chapter 5: Healthy men and women completed three time-to-exhaustion (TTE) tests and quadriceps muscle fatigue was measured after each.  The first TTE served as a control and during the 2nd and 3rd either a hyperoxic mixture was inspired or the work of breathing was lowered. Conclusion The oxygen cost of breathing can be consistently and reproducibly measured in healthy women when the work of breathing during exercise is precisely matched (Chapter 2).  At ventilations above ~55 l min-1, women have a greater oxygen cost of exercise hyperpnea.  During intense exercise, the oxygen uptake of the respiratory muscles in women represents a greater fraction of total oxygen uptake (Chapter 3).  A proportional assist ventilator can reduce the work of breathing to <70% of control values while maintaining spontaneous breathing patterns.  The degree of unloading can be altered, but not in a precise manner (Chapter 4).  Hypoxemia equally influences the development of quadriceps fatigue in both sexes.  Conversely, due to their higher oxygen cost of breathing, manipulating the work of breathing has a greater influence on quadriceps fatigue in women.  The collection of these studies indicates that the normally occurring iii  work of breathing during exercise influences the integrated response to exercise to a greater degree in healthy women. iv  Preface A version of Chapter 1 has been previously published as: Sheel, A.W., Dominelli, P.B., Mogat-Seon, Y. (2016) Revisiting dysanapsis: sex-based differences in airways and the mechanics of breathing during exercise. Exp Physiol. 101.2: 213-218. Author contributions: A.W.S, P.B.D and Y.M.-S contributed to the concepts described within the manuscript and drafting of the article.  Permission has been granted for use: Licence # 3952741254075 (Sept 19th 2016)  The project presented in Chapter 2 received ethical approval from the UBC Clinical Ethics Board (Certificate #H12-01260). A version of Chapter 2 has been previously published as: Dominelli, P.B., Render, J.N., Molgat-Seon, Y., Foster, G.E., Sheel, A.W. (2014) Precise Mimicking of Exercise Hyperpnea To Investigate The Oxygen Cost of Breathing. Respir Physiol Neurobiol 201: 15-23. Conception of study: P.B.D., J.N.R., G.E.F. and A.W.S. Design of experiment: P.B.D., J.N.R., G.E.F. and A.W.S. Data collection, analysis and interpretation and drafting of the article: P.B.D., J.N.R., Y.M.-S, G.E.F. and A.W.S.  Permission has been granted for use: Licence # 3952741458418 (Sept 19th 2016)  The project presented in Chapter 3 received ethical approval from the UBC Clinical Ethics Board (Certificate #H12-01260). A version of Chapter 3 has been previously published as: Dominelli, P.B., Render, J.N., Molgat-Seon, Y., Foster, G.E., Romer, L.M., Sheel, A.W. (2015) Oxygen Cost of Exercise Hyperpnoea is Greater in Women Compared With Men. J Physiol 593.8: 1967-1979.  Author contributions: Conception of study: P.B.D., J.N.R., G.E.F. and A.W.S. Design of experiment: P.B.D., J.N.R., G.E.F. and A.W.S. Data collection, analysis and interpretation and drafting of the article: P.B.D., J.N.R., Y.M.-S, G.E.F., L.M.R. and A.W.S.  Permission has been granted for use: Licence # 3952740995257 (Sept 19th 2016)  The project presented in Chapter 4 received ethical approval from the UBC Clinical Ethics Board (Certificate #H13-00953, H15-01393). A version of Chapter 4 has been v  previously published as: Dominelli, P.B., Henderson, W.H., Sheel, A.W. (2016) . A Proportional assist ventilator to unload respiratory muscles experimentally during exercise in humans. Exp Physiol 101.6: 754-767. Author contributions:  P.B.D,W.R.H. and A.W.S. all contributed to the conception and design, data collection and analysis, and drafting of the manuscript. Permission has been granted for use: Licence # 3952741162906 (Sept 19th 2016).  The project presented in Chapter 5 received ethical approval from the UBC Clinical Ethics Board (Certificate # H13-00953).    vi  Table of contents Abstract ............................................................................................................................... ii Preface ................................................................................................................................ iv Table of contents ................................................................................................................ vi List of tables ..................................................................................................................... viii List of figures ..................................................................................................................... ix List of symbols, abbreviations and nomenclature .............................................................. xi Acknowledgements .......................................................................................................... xiii CHAPTER ONE: INTRODUCTION ..................................................................................1 1.1 Evidence for sex-based differences in human airways ..............................................1 1.2 Mechanical ventilatory constraints ............................................................................2 1.3 Respiratory muscle energetics ...................................................................................4 1.4 Purpose .......................................................................................................................6 1.5 Research questions .....................................................................................................6 1.6 Hypothesis .................................................................................................................6 CHAPTER TWO: PRECISE MIMICKING OF EXERCISE HYPERPNEA TO INVESTIGATE THE OXYGEN COST OF BREATHING ......................................8 2.1 Introduction ................................................................................................................8 2.2 Materials and methods ...............................................................................................9 2.3 Results ......................................................................................................................17 2.4 Discussion ................................................................................................................22 CHAPTER THREE: OXYGEN COST OF EXERCISE HYPERPNOEA IS GREATER IN WOMEN COMPARED WITH MEN ..............................................31 3.1 Introduction ..............................................................................................................31 3.2 Materials and methods .............................................................................................33 3.3 Results ......................................................................................................................40 3.4 Discussion ................................................................................................................45 CHAPTER FOUR: A PROPORTIONAL ASSIST VENTILATOR TO EXPERIMENTALLY UNLOAD RESPIRATORY MUSCLES DURING EXERCISE IN HUMANS. .......................................................................................59 4.1 Introduction ..............................................................................................................59 4.2 Material and methods ...............................................................................................62 4.3 Results and discussion .............................................................................................73 CHAPTER FIVE: REDUCING THE WORK OF BREATHING DURING INTENSE EXERCISE ATTENUATES LOCOMOTOR FATIGUE TO A GREATER EXTENT IN WOMEN .............................................................................................86 5.1 Introduction ..............................................................................................................86 5.2 Methods ...................................................................................................................87 5.3 Results ......................................................................................................................95 5.4 Discussion ..............................................................................................................103 vii  CHAPTER SIX: CONCLUSIONS ..................................................................................113 6.1 Overall summary ....................................................................................................113 6.2 Future directions ....................................................................................................114 6.3 Conclusion .............................................................................................................115 REFERENCES ................................................................................................................117 viii  List of tables Table 1. Improvement in respiratory variables between the familiarization (Day 2) and experimental days (Day 3,4).  Shown is the average % deviation from the target value. ............................................................................................................... 14 Table 2. Mimic ventilation, work of breathing and oxygen cost performed at different fractions of maximal exercise ventilation for men (subject 1-7) and women (subjects 8-13).  Values are mean ± coefficient of variation. ................................... 21 Table 3. Anthropometric and spirometric values. ............................................................. 33 Table 4. Cardiorespiratory values at maximal exercise. ................................................... 34 Table 5. Cardiorespiratory variables during voluntary hyperpnoea at different percentages of maximal exercise ventilation. ........................................................... 49 Table 6. Cardiorespiratory variables during voluntary hyperpnoea at different percentages of maximal exercise ventilation for the EFL (n=10, 50% men) and NEFL group (n=8, 50% men). .................................................................................. 54 Table 7. Baseline variables and maximal exercise data .................................................... 89 Table 8. Cardiorespiratory and quadriceps fatigue variables during the last 50% of the three TTE trials. ........................................................................................................ 99 Table 9. Arterial blood gases variables at baseline and near exercise termination. ........ 101 Table 10. Repeated control TTE trials. ........................................................................... 103  ix  List of figures Figure 1. Schematic overview of the set-up and apparatus for estimating the oxygen cost of breathing. ....................................................................................................... 12 Figure 2. Identity plots correlating values obtained during the initial exercise and those during resting hyperpnea on experimental days (Day 3,4). ............................. 17 Figure 3. Ten second averages of raw data from a female subject during two mimicking trials. ....................................................................................................... 19 Figure 4. Ten second averages of the work of breathing throughout two mimicking trials for a female subject. ......................................................................................... 20 Figure 5. Example of inaccurately high V˙  O2RM  values due to excessive work of breathing. .................................................................................................................. 26 Figure 6. Oesophageal pressure-volume loops for a representative male subject during exercise (Pane A-D) and voluntary hyperpnoea (Panel E-H). .................................. 36 Figure 7. Relationship between work of breathing and minute ventilation during the voluntary hyperpnoea ................................................................................................ 40 Figure 8. Relationship between elastic (Panel A) or resistive (Panel B) work of breathing and minute ventilation .............................................................................. 42 Figure 9. Panel A: average oxygen uptake for each stage of voluntary hyperpnoea performed by each subject.  Panel B: regression lines for men and women performing voluntary hyperpnoea trials.  The star, vertical line and arrow indicate that women have a significantly higher V˙  O2RM above a ventilation of ~55 l min-1.  Regression was fitted using the average of each subject’s constants.  Panel C: absolute V˙  O2RM at different percentages of maximal ventilation.  Men had significantly greater ventilations at every comparison (see also Table 3).  Panel D: V˙  O2RM as a percentage of whole-body oxygen uptake at different percentages of maximal ventilation. ......................................................................... 43 Figure 10. Box and whisker plot, showing individual subject data and group mean ± SE for V˙  O2RM as a percentage of whole-body oxygen uptake at maximal exercise in women and men. ................................................................................................... 45 Figure 11. Oxygen uptake of the respiratory muscles in absolute (Panel A) and relative units (Panel B) at different WOB for each subject performing each trial. ... 47 Figure 12. Composite average maximal expiratory flow-volume (MEFV) curves for subjects displaying expiratory flow limitation (EFL) (Panel A) and those with no expiratory flow limitation (NEFL) (Panel C). .......................................................... 52 x  Figure 13. Schematic representation of the proportional assist ventilator. ....................... 65 Figure 14. Raw traces of flow (Panel A), mouth pressure (Panel B) and power consumption by the motor driving the piston (Panel C) from the breathing simulator. .................................................................................................................. 69 Figure 15. Raw respiratory pressures and flow from a subject breathing on the apparatus with the PAV assisting (left of the solid vertical line) and not providing assist (right of the solid vertical line). ...................................................... 74 Figure 16. Oesophageal pressure throughout an inspiration during assisted breathing (PAV) and the control condition for a single subject exercising at different intensities on a cycle ergometer. ............................................................................... 76 Figure 17. Panel A, work of breathing values from integrating oesophageal pressure-volume loops at different ventilation.  All data is from a single male subject completing different exercise tests within 2 months.  Panel B, work of breathing from pressure-time products for three constant load time-to-exhaustions exercise tests performed for identical time and work in a single female subject. ................... 78 Figure 18. Quadriceps twitch force (Panels A, B) and M-wave amplitude (Panel C, D) during magnetic stimulation of the femoral nerve at different stimulator outputs for men (Panel A, C) and women (Panel B, D). ....................................................... 96 Figure 19. Group mean arterial blood gas and oesophageal temperature throughout the control (Day 2) time-to-exhaustion test. ............................................................. 98 Figure 20. Changes in quadriceps twitch force across time and for the different conditions and each sex. ......................................................................................... 102 Figure 21. Relationship between the degree of quadriceps fatigue attenuation and the nadir oxyhaemoglobin saturation (FO2Hb) for the control trial. ............................ 105 Figure 22. Work of breathing for the control trial and the proportional assist ventilation trial.  Regression lines in Panel A are redrawn from maximal exercise.  Small symbols in Panel A and B are individual data while large symbols are group averages.  In Panel C, “Fatigue” is the % difference in quadriceps fatigue between control and PAV trials. ............................................... 108    xi  List of symbols, abbreviations and nomenclature Symbol Definition A-aDO2 Alveolar to arterial oxygen difference CaO2 Arterial oxygen content E Elastance EELV End-expiratory lung volume EIAH Exercise-induced arterial hypoxemia EILV End-inspiratory lung volume EFL Expiratory flow limitation EMG Electromyograms Expval Expiratory valve FIO2 Inspired oxygen fraction FICO2 Inspired carbon dioxide fraction FO2Hb Fraction of oxyhemoglobin fR Breathing frequency FVC Forced vital capacity IC Inspiratory capacity Inspval Inspiratory valve MEFV Maximal expiratory flow volume MVC Maximal voluntary contraction PETCO2 End-tidal carbon dioxide tension PaO2 Arterial oxygen tension PAO2 Alveolar oxygen tension PaCO2 Arterial carbon dioxide tension PAV Proportional assist ventilator Paw Airway pressure PDI Transdiaphragmatic pressure PES Esophageal pressure PGA Gastric pressure PM Mouth pressure Pmus Muscle pressure PTP Pressure-time product PVR Pulmonary vascular resistance R Resistance SaO2 Arterial oxygen saturation Solval Solenoid valve TI Inspiratory time TTE Time to exhaustion V˙   Flow V Volume V˙  A Alveolar minute ventilation V˙  CO2 Carbon dioxide output V˙  E Expired minute ventilation V˙  ECap Ventilatory capacity xii  V˙  Emax Maximal expired minute ventilation V˙  O2 Oxygen uptake V˙  O2max Maximum oxygen uptake V˙  O2RM Oxygen uptake of the respiratory muscles V˙  O2tot Whole-body oxygen uptake VT Tidal volume WOB Work of breathing xiii  Acknowledgements I would like to acknowledge my thesis supervisor, Dr Bill Sheel, and the members of my supervisory committee, Drs Glen Foster, Lee Romer, and Michael Koehle, for their guidance and scholarly support throughout my studies.  I would also like to thank members of the HIP lab for their patience and assistance with data collection.  I am also thankful to the physicians (Drs: Henderson, Dominelli, Griesdale, Sekon) who performed the medical procedures throughout my graduate studies.  Finally, I would like to thank Dr. Robert Parson for his guidance and wisdom in developing the PAV.  1  Chapter One: Introduction  1.1 Evidence for sex-based differences in human airways  The concept that individuals with large lungs do not necessarily have larger-diameter airways than do persons with small lungs was first described as ‘dysanapsis’ (48).  Here, the term was used to reflect the physiological variation in the geometry of the tracheobronchial tree and lung parenchyma resulting from different patterns of growth.  As a corollary to the initial observation of dysanapsis, the association between airway and lung size in adult women and men was determined by quantifying the ratio between a measurement sensitive to lung size (vital capacity) and a measurement sensitive to airway size [expired flow, static recoil; (90)].  The results of this analysis suggest that healthy adult men have airways that are ~17% larger in diameter than are the airways of women.  Other methods have yielded similar findings; for example ,measurement of acoustic reflectance has shown that the tracheal cross-sectional area is 29% smaller in women compared with men matched for total lung capacity (86). A limitation of the above-mentioned studies is that they provide an index of airway size or only assess the tracheal ‘region’ rather than a series of discrete anatomical points.  Using high-resolution computed tomography in older ex-smokers, we have shown that the luminal areas of the larger and central airways are 14–31% smaller in women relative to men even when matched for lung size [Fig. 2Ain (120)].  Observations of sex differences in airway anatomy are important when considering the principles of airflow.  The main sites of resistance (~80%) are the larger airways, whereas the smaller airways contribute <20%, and airflow resistance is inversely proportional to radius to the fourth power.  These collective observations (86, 90, 120) lead us to predict that during dynamic exercise, when ventilation and flows are high, a woman matched for lung size to a man would have higher airway resistance and more turbulent airflow.  Moreover, a high  2  airway resistance would manifest as a higher propensity for mechanical ventilatory constraints and greater mechanical and metabolic cost of ventilation.  1.2 Mechanical ventilatory constraints Even when matched for height, women have significantly smaller lung volumes than men, which is likely to be related to a smaller total number of alveoli, as demonstrated by post-mortem examination of lungs obtained from boys and girls [6 weeks to 14 years old; (130)].  Additionally, sex-based differences in airway size result in women having lower maximal expiratory flows for a given lung volume.  Smaller lung volumes and lower maximal expiratory flows result in women having a relatively reduced ventilatory capacity when compared with men, which is reflected in the size of their respective maximal expiratory flow–volume envelopes.  During dynamic whole-body exercise, ventilation (V˙  E) increases progressively as metabolic rate rises.  However, the respiratory system possesses a substantial reserve for increases in V˙  E and is generally considered ‘overbuilt’ for the ventilatory demand imposed by maximal exercise (28).  Nevertheless, young male endurance-trained athletes capable of attaining high metabolic rates often reach the mechanical limits of their respiratory system (75). Given that women have a lower ventilatory capacity than men, it is reasonable to assume that women may be more susceptible to mechanical ventilatory constraints (53, 87).  One method of assessing whether an individual has reached their ventilatory limits is by determining the presence of expiratory flow limitation (EFL).  We have shown that endurance-trained women experience EFL more frequently and at a lower equivalent V˙  E compared with endurance-trained men (53).  Expiratory flow limitation can be avoided by altering operational lung volumes and/or breathing patterns.  3  Increasing the end-expiratory lung volume while maintaining the same tidal volume can alleviate EFL by allowing higher expiratory flows, which are available at higher lung volumes.  Likewise, altering the breathing pattern by decreasing tidal volume and increasing breathing frequency maintains V˙  E without having to encroach on inspiratory reserve or reaching maximal expiratory flow.  Although both these compensatory strategies are beneficial in the context of EFL, they carry inherent physiological consequences.  When operational lung volumes are increased, breathing occurs on the higher, less-compliant portion of the pressure–volume curve, thereby altering the length–tension relationship of the diaphragm and increasing the respiratory muscle work and neural inspiratory drive required to generate a given V˙  E.  Adopting a tachypneoic breathing pattern increases the ratio of dead space to tidal volume, which decreases alveolar ventilation (V˙  A) for a given V˙  E.  Without an adequate compensatory increase in V˙  E, relative alveolar hypoventilation leads to a decrease in arterial oxygen tension and an increase in arterial carbon dioxide tension. Indeed, there is evidence to support the suggestion that reaching the mechanical ventilatory limits of the respiratory system may lead to or exacerbate exercise-induced arterial hypoxaemia (EIAH;(29, 33)). In the case of EFL, the inability to increase V˙  E adequately in proportion to metabolic load leads directly to EIAH or constrains V˙  E to a rate that is insufficient to reverse the attendant hypoxaemia completely.  We have shown that experimentally increasing ventilatory capacity in individuals with EFL by using a helium–oxygen inspirate abolishes EFL and partly relieves EIAH (33).  Although our data suggest that mechanical ventilatory constraints can play a contributory role in the genesis of EIAH, it is important to note that the cause of EIAH is multifactorial and is unlikely to be the result of a single mechanism, because ventilation–perfusion inequality and failure in alveolar end-capillary diffusion equilibration are known mechanisms.    4  1.3 Respiratory muscle energetics At rest, the energetic cost of maintaining adequate V˙  A is minimal and presents little challenge for the respiratory muscles.  However, during whole-body exercise, V˙  A must increase substantially and as a consequence the work of breathing (WOB) rises in an exponential fashion relative to V˙  E (102).  Some of the first data to suggest that women have a higher WOB came from investigating both sexes during cycle exercise (134).  It was observed that although men had a significantly greater V˙  E at maximal oxygen consumption (V˙  O2max), women exhibited greater ventilatory work, as evidenced by a higher oesophageal tension–time index.  The authors concluded that sex differences in WOB were the result of differences in physical size, absolute lung function and breathing patterns; with the average women being shorter, having smaller lung volume and breathing with a relatively greater frequency. Recently, we extended these findings to endurance trained men and women during cycle exercise, where at V˙  E >65 l min−1 women had a significantly greater WOB (53).  As V˙  E increased, the difference became amplified, with the women’s WOB being twice that of the men at V˙  E >90 l min−1.  However, when the WOB was compared at different percentages of V˙  O2max, no sex differences were present, despite men having greater V˙  E.  When the WOB was analysed using Campbell diagrams to partition the WOB into inspiratory–expiratory and elastic–resistive components, further sex differences emerged. At values of V˙  E of 50, 75 and 100 l min−1, there were no sex differences in the elastic components of WOB, but women had greater inspiratory resistive work (51).  We attribute the greater resistive work in women to be indicative of smaller airways.  The lack of sex differences in the elastic WOB is consistent with findings of similar intrinsic  5  elastic tissue between men and women, with no apparent difference in static recoil pressures (25). An important limitation to our previous studies (51, 53) is the exclusive use of endurance-trained subjects. Perhaps prolonged physical training has resulted in alterations in lung function that manifest differently as a function of sex?  Or is the reported sex difference in total and resistive work merely a function of the high V˙  E of endurance-trained subjects resulting in higher resistances?  We have recently addressed this question by investigating the WOB in both highly trained and less trained men and women with a range of values for V˙  O2max and maximal V˙  E (37).  We created a mathematical model that accounts for viscoelastic and resistive components of the total WOB and independently compares the effects of sex (37).  We found that the high WOB during exercise in women is the result of greater resistive work rather than viscoelastic work, which is explained by sex.  In the same cohort, we found that the dysanapsis ratio was statistically similar between men and women, which is explained by the significantly smaller vital capacity in women.  In order to account for the effect of absolute lung volumes on the dysanapsis ratio, we compared the sexes with an analysis of covariance procedure and found that when vital capacity is corrected for, the adjusted mean dysanapsis ratio is statistically lower in women.  Our findings suggest that innate sex differences exist in human airways, which result in significant male–female differences in the resistive and total WOB during exercise in health.  However, while this study did utilize non-endurance trained subjects, both the men and women are still more fit than a typical population.  To our knowledge, sex-difference in WOB during exercise has yet to be identified in population representative of a true norm with modest a V˙  O2max. When assessing sex differences in WOB during exercise, there are two important considerations.  Firstly, one must consider the effect of absolute size when interpreting sex differences.  Although some of our findings are independent of body and lung size,  6  often sex differences can be attributed simply to scale.  Secondly, the WOB should be acknowledged to be a best estimate, with some components of respiratory work not taken into account. When integrating pressure–volume loops, the real WOB can be underestimated by more than 25% (47).  Specifically, the work done in distorting the chest wall and stabilizing the abdomen is not taken into account (49), nor is the work done overcoming the inertia of the respiratory system.  To date, it is unknown whether there are any sex-based differences in the aforementioned factors.  1.4 Purpose The purpose of this thesis was to investigate sex differences in the integrated response to high respiratory muscle work during intense exercise.  This was accomplished by developing two methodologies to answer two primary research questions. 1.5 Research questions 1.   For a given ventilation, do women have a greater absolute V˙  O2RM and does this represents a larger percentage of whole-body V˙  O2 compared with men? 2. To what extent does eliminating EIAH attenuate quadriceps muscle fatigue in both sexes?  Will reducing WOB during intense exercise attenuate quadriceps muscle fatigue similarly in both sexes 1.6 Hypothesis 1. We hypothesized that at submaximal and maximal ventilations where the WOB is greater compared to men, women have a greater V˙  O2RM and this constitutes a larger proportion of whole-body oxygen uptake  7  2.  We hypothesized that, regardless of sex, those subjects who develop the lowest SaO2 during exercise will have the greatest attenuation of quadriceps fatigue when EIAH is prevented.  We further hypothesized that when WOB is reduced, women will show a greater attenuation of quadriceps fatigue.  8  Chapter Two: Precise mimicking of exercise hyperpnea to investigate the oxygen cost of breathing  2.1 Introduction With increasing exercise intensity, the higher metabolic demands necessitate a corresponding increase in expired minute ventilation (V˙  E).  As V˙  E rises, the mechanical work of breathing (WOB) also increases and does so in a hyperbolic fashion (103).  Work is linearly related to oxygen uptake (10) therefore, exercise-induced increases in the WOB require a parallel increase in the rate of respiratory muscle oxygen consumption (V˙  O2RM ).  Since the WOB- V˙  E relationship is non-linear (102), it stands to reason that the relation between V˙  O2RM - V˙  E would follow a similar function.  Thus, in order to fully characterize the oxygen cost of exercise hyperpnea it is necessary to use a wide range of V˙  E values.   There have been numerous attempts to quantify the V˙  O2RM using different methods and the results have varied considerably (137).  Not only do the overall techniques vary, but also the duration, level of ventilation and specific population studied (i.e. healthy or clinical) are varied.  Not surprisingly, the myriad of methodologies employed have resulted in considerable variation in V˙  O2RM between studies.  For example, at a V˙  E of 100 l min-1 the reported V˙  O2RM can vary from 50-320 ml min-1(111).  Some investigators have had subjects breathe at fractions of maximal voluntary ventilation which often results in hyperflation and tachypneic breathing resulting in the WOB being substantially higher (20-300% greater) than what is commonly observed during “natural” breathing patterns (77).  The linear work- V˙  O2 relationship requires that WOB be controlled, otherwise poor estimates of V˙  O2RM are made (2).  During voluntary hyperpnea, breathing becomes conscious and tidal volume (VT), breathing frequency (fR), inspiratory time and respiratory muscle recruitment may differ from patterns observed during spontaneous breathing during exercise (103).  The potential result from the  9  unstructured conscious breathing is unnatural and inefficient patterns contributing to greater WOB.  Thus, in order to accurately assess V˙  O2RM it is crucial that all aspects of exercise hyperpnea are carefully reproduced in order to ensure inspiratory and expiratory work are identical. Most methods used to estimate V˙  O2RM result in a different end-tidal CO2 (PETCO2), compared to what would normally be observed during exercise.  Changes in PETCO2 are used as a surrogate for arterial PCO2, which is known to affect pulmonary and systemic vasculature (26, 41, 78).  As such, voluntary mimicking of exercise hyperpnea with PETCO2 maintained pressures similar to each exercise stage is necessary in order to provide the best estimate of V˙  O2RM .    Accordingly, the purpose of this study was to develop an improved methodology for estimating V˙  O2RM by voluntary hyperpnea and to determine its ability to reproduce exercise breathing patterns and mechanics.  The current paper describes a new methodology for providing visual on-line feedback of many exercise related parameters and the maintenance of end-tidal gases.  We hypothesized that with extensive feedback aimed at accurately mimicking exercise breathing pattern and WOB, we would be able to consistently estimate the V˙  O2RM .   2.2 Materials and methods Subject characteristics. Thirteen young subjects (7 male) participated after providing written informed consent.  All procedures were approved by the Clinical Research Ethics Board at the University of British Columbia.  Subjects were healthy nonsmokers, had no history of cardiopulmonary diseases, had normal pulmonary function (all values >90% predicted (127)) and regularly engaged in physical activity.     10  Overview. Subjects visited the laboratory on four occasions (Days 1-4).  The second testing day occurred a minimum of 48 hours after the first testing day, while the other visits were separated by 24 hours to 1 week.  Baseline pulmonary function tests and a graded maximal exercise test on a cycle ergometer were completed on Day 1.  The second day served to familiarize the subjects with the voluntary hyperpnea protocol.  On Days 3 and 4 subjects mimicked their exercise breathing patterns while resting in their cycling position.   Subjects were instrumented with esophageal and gastric balloon catheters on the 1st, 3rd and 4th testing day.  Maximal exercise test (Day 1.) Subjects completed an incremental cycle exercise test to exhaustion using a stepwise protocol on an electromagnetically braked cycle ergometer (Excalibur Sport, Lode, the Netherlands).  Prior to the maximal test, the bike and handle bars were adjusted and a 10-minute self-selected warm-up was completed.  Exercise started at 80W for women and 120 W for men, and both increased in 20 W increments every two minutes.  The test was terminated when the subjects could no longer maintain a cadence of >60 rpm despite verbal encouragement.  Data acquisition. During all tests, raw data (flow, pressures, end-tidal, inspired gas fractions and mixed-expired gases) were recorded continuously at 200Hz using a 16-channel analog-to-digital data acquisition system (PowerLab/16SP model ML 795, ADInstruments, Colorado Springs, CO, USA) and stored on a computer for subsequent analysis. Ventilatory and mixed expired metabolic variables were gathered using a customized metabolic cart consisting of independent inspired and expired pneumotachographs (model 3818, Hans Rudolph, Kansas City, MO, USA), and two pairs of independently calibrated O2 and CO2 analyzers (SA/II and CD-3A respectively;  11  Applied Electrochemistry, Pittsburg, PA).  Inspired fractions of O2 (FIO2) and CO2 (FICO2) were determined by sampling gas on the inspired circuit immediately before the inspired pneumotachograph and expired gas fractions were determined by sampling from a mixing chamber distal to the expired pneumotachograph (Figure 1).  During exercise (Day 1) subjects inspired room air.  End-tidal CO2 was sampled at the mouth through a port in the mouthpiece of a two-way non-rebreathing valve (Hans Rudolph 2700B, Hans Rudolph, Kansas City, MO, USA) and connected to a calibrated CO2 analyzer (Vacumed model 17630, Ventura, CA).  The inspiratory and expiratory portions of the two-way non-rebreathing valve were connected to the pneumotachographs via wide bore tubing.  At the highest relevant flows (~9 l sec-1) the difference in apparatus resistance between the exercise trials and hyperpnea trials was 0.1 cmH2O l-1 sec-1. Mouth pressure (PM) was sampled through a second port in the mouthpiece while esophageal (PES) and gastric (PGA) pressures were measured by balloon-tipped catheters (no. 47-9005, Ackrad Laboratory, Cranford, NJ, USA).  The balloon catheters were placed after application of a topical anesthetic and were passed through the nose and positioned according to previously described techniques (93). Pressures were measured by a piezoelectric pressure transducer (Raytech Instruments, Vancouver, BC, CA), and calibrated by a digital manometer.  Transdiaphragmatic pressure (PDI) and transpulmonary pressure (PTP) were calculated from the difference of PGA and PES, and PES and PM, respectively. Operational lung volumes were determined at rest, each exercise stage and each hyperpnea trial by inspiratory capacity (IC) maneuver.  If the IC maneuver was not performed adequately, another IC maneuver was prompted before the end of the stage or trial.  End expiratory lung volume (EELV) was calculated by subtracting the IC volume from the forced vital capacity (FVC) volume.  End-inspiratory lung volume (EILV) was calculated as the sum of VT and EELV.  Maximal expiratory flow-volume curves, were  12  developed using pre and post exercise FVCs (to account for bronchodilation) and with different efforts (to account for thoracic gas compression) while the subject was seated in   Figure 1. Schematic overview of the set-up and apparatus for estimating the oxygen cost of breathing.   Illustration is not to scale.  PT, pneumotachograph the cycling position on the bicycle ergometer; methods for collection have been described previously (35, 50).  A heart-rate monitor (S610i, Polar Electro, Kempele, Finland) was worn on the chest, and heart rate was recorded every minute.  Blood pressure was obtained with an automated blood pressure cuff (UA-767, Life Source).  Exercise analysis. Cardio-respiratory variables were averaged over the last 30 seconds of each exercise stage and at maximal intensity.  Composite average flow-volume and pressure-volume loops were constructed by signal averaging the breaths within the 30 second average.  Errant breaths or erroneous noise such as coughing or swallowing were individually excluded before the breaths were averaged.  At every stage, flow-volume loops were placed within the maximal expiratory flow-volume curve according to EELV as determined by the IC maneuver.  Using the composite average pressure-volume loops we determined the energetic WOB and divided the work into the constituent elastic and resistive components using modified Campbell diagrams (40).  All analysis was  13  performed using customized software (GNARx, developed with LabView 2013, National Instruments, Austin, Tx, USA)  Familiarization visit (Day 2). Day 2 served to familiarize the subjects with the mimicking procedures and the extensive feedback provided (see below).  Between 2-5 trials of each workload were performed to allow sufficient practice and to improve accuracy on following testing days.  Balloon catheters were not utilized this day nor was data used for V˙  O2RM estimates.  All other procedures and analyses were identical to experimental days described below.  Voluntary hyperpnea to estimate V˙  O2RM (Day 3 and 4). Subjects returned after Day 2 to mimic, at rest the ventilatory variables from multiple exercise workloads, including maximal intensity.  Subjects were asked to refrain from caffeine and exercise before the final two days of testing.  Balloon catheters were inserted and placed, after which the subjects sat quietly on the mouthpiece for 10-20 minutes to obtain resting metabolic data.  As resting V˙  O2 can vary throughout the day (106), multiple resting values were recorded between workloads and trials.  Subjects mimicked exercise hyperpnea corresponding to their maximal workload as well as two additional exercise workloads (50 and 75% V˙  E).  A total of 3-5 trials, based on time and accuracy, were complete for each exercise workload.  During all trials, great care was taken to ensure the subjects were in a similar position on the cycle ergometer.  Mimicking trials lasted 5 minutes each, to ensure steady state and adequate replication of exercise parameters and the IC maneuver was performed during the last 15 seconds.  Data from a 60-second period of hyperpnea immediately prior to the IC maneuver was averaged and used to calculate the V˙  O2RM .  Workloads were randomized, however, all trials for one workload were performed in succession.  The expired gas mixing chamber  14  was vented by a fan immediately prior to and after each mimicking trial.  Each trial was separated by 10-15 minutes of rest while heart rate and blood pressure were monitored to ensure they had returned to baseline.  Inspired oxygen fraction was always ~21% while FICO2 was adjusted to ensure PETCO2 was similar to each respective exercise stage, see “Mixed Inspired Gases.” Feedback to accurately mimic exercise breathing parameters was as follows.  A metronome was set to match fR and inspiratory/expiratory duty cycle.  Tidal volume was adjusted via verbal feedback from the investigators: “take larger” or “smaller breaths,” accordingly.  Target flow-volume and pressure-volume loops were displayed on a computer screen directly in front of the cycle ergometer (described below).  The investigators also monitored the subject’s body position to ensure it was similar to their cycling position.  In addition to verbal feedback, occasionally a towel was lightly wrapped around the subjects’ stomach to provide proprioceptive feedback which reinforces diaphragm activation during the hyperpnea.  All other cardio-respiratory and metabolic variables were available to the investigators (not the subjects) in real-time.  Table 1. Improvement in respiratory variables between the familiarization (Day 2) and experimental days (Day 3,4).  Shown is the average % deviation from the target value.   Familiarization Experimental VT (%) 5.8±0.7 4.2±0.5* range 1.7-10.4 1.6±6.7 fR (%) 2.6±0.5 2.0±0.3 range 0.8-5.2 1.1-4.1 V˙  E (%) 5.4±0.9 3.7±0.4* range 1.5-11.1 1.4-5.6 V˙   (%) 17.7±1.0 15.0±0.5* range 15.3-25.0 12.0-17.0 Definition of abbreviations: VT, tidal volume; fR, breathing frequency; V˙  E, expired minute ventilation. V˙   ; inspiratory and expiratory flow throughout flow-volume loop. * significantly different from familiarization day.  Values are means ± SE.   15  On-line feedback of flow-volume and pressure-volume loops. The flow signal from the inspired and expired circuits, PM, PES and PGA were integrated by custom software to create a real-time display of inspiratory and expiratory flow, volume and pressures (OPVB, developed in LabView 2013, National Instruments, Austin, Tx).  Flow-volume and pressure-volume loops were displayed in real-time and overlaid on a fixed target exercise loop for the given stage of exercise being replicated.  Tracings were displayed on a computer monitor directly in front of the subject (insert on Figure 1).  Flow-volume loops were always presented to the subject on one panel while the second panel could be switched between: PM, PES, PGA, PTP and PDI –volume loops; primarily PES was used.  Subjects were instructed to alter the force of their breath and the volume to match the target flow-volume/pressure-volume loop.  Verbal coaching was provided to make breaths smoother, faster and to ensure the diaphragm or accessory muscles were appropriately recruited to better match the pressure-volume loop.  The ratio of diaphragmatic to esophageal pressure was used to gauge diaphragm vs. accessory muscle activation.  End-expiratory esophageal pressure was also used as a surrogate to ensure EELV was similar between exercise and voluntary hyperpnea.  Thirty seconds of continuous traces were visible to the subject at all times, with the initial 15 seconds getting cleared automatically.   To determine the % deviation of inspiratory and expiratory flow (Table 1) we compared averaged exercise flow-volume with averaged familiarization and experimental flow-volume loops.  Each loop was divided into 202 points (101 inspiratory) and the absolute % deviation from the exercise loop was determined for the familiarization and experimental days on a point-by-point basis.  Values presented in Table 1 are the average deviation throughout the whole flow-volume loop.   16  Mixed inspired gases. To maintain PETCO2 at a level identical to the exercise stage, customized software (Gas Mixer Alpha, developed in LabView 2013, National Instruments, Austin, Tx) was used to create mixed inspired gas with ~2-6% FICO2 and ~21% FIO2.  The software used inputs from the alveolar gas and alveolar ventilation equation to determine the inspired gas fractions needed to obtain a desired .  To obtain the gas fractions, tanks of medical grade compressed N2, O2 and CO2 were each connected to normally-closed single movement solenoid valves.  The solenoid valves were connected to digital relays which, when triggered by a digital signal, powered the opening and closing of the valve.  The digital signal was provided to each relay from a data acquisition board (USB-6229 BNC, National Instruments, Austin, Tx, USA).  Gas flow through each valve (for a given driving pressure) was determined prior to each experimental day, thus allowing for calculation of the duration each valve should stay open during creation of the mixed gas.  As N2 was the primary gas, this valve remained open the entire filling time, whereas the O2 and CO2 valves would open and close at a calculated frequency to improve the mixing of gas.  All gases were contributing to the volume throughout the whole creation of the inspirate to ensure consistent mixing and homogeneity of the final product.  Gases were further mixed in a humidification chamber before filling an apparatus consisting of one to three 200 l meteorological balloons (model 1197, Vacumed, Ventura, CA) connected to the inspired circuit (Figure 1).  Depending on the target ventilation of a given trial, the mixed inspired gases could be routed to fill a combination of 1-3 balloons.  Inspired gas was made immediately prior to the trial, which began immediately upon cessation of balloon filling, thus minimizing any diffusion of gases.  Measured concentrations of inspired gases were used in the calculation of  and . The absolute variance about the mean for inspired gas throughout a trial was negligible (0.001-0.02% for O2 and 0.007-0.01% for CO2).    17   Figure 2. Identity plots correlating values obtained during the initial exercise and those during resting hyperpnea on experimental days (Day 3,4).   Each point represents all trials from each stage for each subject.  fR, breathing frequency; V˙  E, expired minute ventilation; EELV, end-expiratory lung volume; PetCO2 end-tidal carbon dioxide pressure; PES, esophageal pressure; WOB, work of breathing.  Statistical analysis. The percent deviation from the target on the familiarization day (Day 2) and the experimental days (Days 3, 4) was compared using dependent students t-test.  Pearson product moment correlation was used to determine the relationship between selected variables.  Significance was set at P<0.05.   2.3 Results Subjects were 26±2 (men: 30±4; women: 23±1) years old, 175±3 cm tall (men: 183±2; women: 165±3), weighed 66±3 kg (men: 74±3; women: 57±2) and had a maximal  18  oxygen consumption (V˙  O2max) of 54±2 ml kg-1 min-1 (men: 60±2; women: 48±3).  Accordingly, while the subjects were not exclusive endurance trained athletes; both sexes would be considered “well trained” compared to population norms.  The percent difference between the target values and the mean over the final minute of each trial for VT and V˙  E was lower on the experimental days (Days 3, 4) (Table 1).  The improvement in VT , V˙  E  and flow was variable between subjects, with some reducing their error by ~5% while others only minimally (<1%).  However, only one subject showed an increase in variance after Day 2, and this was only for V˙  E and was minor (0.8%).  The ability to voluntarily mimic exercise hyperpnea is shown in Figure 2.  Breathing frequency,V˙  E  (Figure 2, panels A, B) and VT (r2=0.99) during mimicking trials were in very close agreement with their respective exercise stages.  There was more variability with the ability to match EELV (panel C) especially at higher volumes with a positive bias, which could be due to the inability to obtain a continuous measurement, and therefore, limited feedback was available to the subjects.  We also performed a Bland-Altman plot with EELV (data not shown) and despite there being a positive bias at high lung volumes, all points were within the 95% interval.  Mimicked PETCO2 were similar to exercise with few exceptions (panel D).  These exceptions occurred when exercise  PETCO2 was slightly elevated (>45mmHg) during some low intensity exercise stages.  In the above cases the experimenters erred towards mild relative hypocapnia, but only similar to exercise stages (40-45 mmHg range).  We were able to consistently replicate the changes in PES and WOB during all exercise stages (Figure 2, panel E, F).   Figure 3 displays data from two trials in a single representative subject mimicking an exercise stage (~75% of V˙  O2max ).  Shown is the temporal change in selected variables for the whole 5 minute trial.  The high V˙  O2 at the beginning of the trial was present in all subjects in every trial.  This was the result of a near instantaneous increase in V˙  E, while  19  equilibrium of alveolar gases, changes in fractions of mixed expired gases and sampling time (transit between sampling site and analyzer) all have an inherent   Figure 3. Ten second averages of raw data from a female subject during two mimicking trials. Gray shaded area represents ±5% of the target value.  The vertical dashed lines demarcate the final minute used for the trial average.  The V˙  O2RM for the first trial was 0.130 l min-1 and 0.125 l min-1 for the second. V˙  O2, oxygen consumption; VT, tidal volume; V˙  E, expired minute ventilation; fR, breathing frequency; PES, esophageal pressure; PetCO2, end-tidal carbon dioxide pressure.  Values are mean±SE. delay. After approximately 1 minute, FEO2  stabilized and the V˙  O2 began to mirror WOB and V˙  E.  Tidal volume, fR and V˙  E, were consistent throughout the entire trial (Figure 3, panel B, C, D).  End-tidal carbon dioxide  pressures were consistent and showed little to no deviation (<1 mmHg) (Figure 3, panel E).  The changes in PES (Figure 3, panel F) along with WOB (Figure 4) required more extensive coaching and took longer to stabilize, but were overall similar to the targets.  The changes in V˙  O2 did not exactly  20  mirror the variations in WOB, due to the inherent delays described above, but was most stable during the final ~1 min when WOB was consistent.  With few exceptions, the matched variables (V˙  E, VT, fR, PETCO2, PES, WOB, etc.) were within 5% of the target for the final minute of the trial, which is where V˙  O2RM calculations were completed.  Table 2 shows mean values and coefficients of variation (CV) for each subject mimicking 50, 75 and 100% of their V˙  Emax.  Except for two stages, all CV for V˙  O2 were <10% and were predominantly ~5%.  There was no difference between men and women in their ability to consistently mimic their exercise parameters  Figure 4. Ten second averages of the work of breathing throughout two mimicking trials for a female subject. Gray shaded area represents ±5% of the target value.  The vertical dashed lines demarcate the final minute used for the trial average.  WOB, work of breathing.  Values are mean ± SE. 21  Table 2. Mimic ventilation, work of breathing and oxygen cost performed at different fractions of maximal exercise ventilation for men (subject 1-7) and women (subjects 8-13).  Values are mean ± coefficient of variation.  50% V˙  Emax 75% V˙  Emax 100% V˙  Emax n V˙  E          (l min-1) WOB (J min-1)            (l min-1) n V˙  E              (l min-1) WOB (J min-1)          (l min-1) n V˙  E          (l min-1) WOB (J min-1)             (l min-1) 1 M 3 63±4.6 74±7.1 0.37±2.3 3 94±4.2 111±8.2 0.40±4.5 4 133±3.5 225±7.6 0.55±1.7 2 M 3 86±1.9 95±11.9 0.42±4.1 3 123±2.0 195±3.5 0.46±1.8 5 164±5.1 396±6.1 0.75±9.8 3 M 5 77±1.5 89±4.1 0.38±3.5 5 119±1.0 174±3.5 0.45±6.1 5 172±1.7 360±11.3 0.70±4.9 4 M 4 111±2.6 126±7.2 0.40±3.2 5 157±1.8 299±9.6 0.70±5.0 3 187±3.5 490±21.9 0.71±15.1 5 M 3 110±1.0 181±7.3 0.47±4.4 5 156±1.9 332±9.8 0.52±3.5 5 191±1.1 664±3.3 0.75±1.6 6 M 4 64±3.2 61±15.7 0.39±3.5 4 108±4.5 132±8.6 0.47±5.7 4 133±4.4 278±22.5 0.60±7.1 7 M 5 93±3.5 96±4.3 0.47±3.8 3 153±1.7 290±12 0.59±2.5 3 180±1.1 868±1.4 0.94±6.3 8 F 4 56±2.4 52±2.8 0.39±3.1 4 81±4.1 135±19.4 0.43±2.6 4 105±4.7 277±14.0 0.84±2.7 9 F 4 65±2.2 89±5.4 0.37±5.6 4 90±1.42 161±10.6 0.52±9.7 4 118±1.9 313±10.0 0.69±7.3 10F 5 46±3.5 51±7.9 0.34±3.2 5 79±3.2 108±4.5 0.40±2.9 5 91±3.2 185±5.9 0.44±4.2 11F 5 66±10.0 70±23.1 0.33±11.0 4 90±4.7 141±14.8 0.41±3.8 4 105±8.0 182±18.8 0.52±6.9 12F 5 49±3.0 47±12.9 0.28±5.0 5 74±1.9 122±8.3 0.37±3.5 5 94±4.6 285±9.3 0.58±4.0 13F 5 56±3.2 46±12.2 0.32±3.7 5 94±1.8 191±11.5 0.46±5.7 5 119±1.5 266±12.4 0.52±2.5 M  86±2.6 103±8.2 0.41±3.5  130±2.4 219±7.9 0.51±4.2  166±2.9 469±10.6 0.71±6.6 F  56±4.0 58±10.7 0.34±5.3  95±2.8 143±11.5 0.43±4.7  104±4.0 252±11.7 0.60±4.6 All  73±3.3 82±9.4 0.38±4.3  109±2.6 184±9.6 0.48±4.4  137±3.4 368±11.1 0.66±5.7 Definition of abbreviations: n, number of trials; V˙  E, expired minute ventilation; WOB, work of breathing; , oxygen consumption; M, male; F, female  22    The importance of accurately mimicking exercise WOB rather than simply V˙  E is shown in Figure 5.  The figure depicts a male subject’s tidal flow-volume loops, esophageal pressure-volume loops and ventilatory variables during mimicking of their V˙  O2max breathing pattern for two trials.  For both trials, the subject’s VT, fR and V˙  E were nearly identical.  However, in one trial, their WOB was substantially higher owing to excessive pressure generated on expiration.  The high pressure was over and above the maximal effective driving pressure, which is wasted effort as no gains in flow occur (i.e. expiratory flow limitation).  The excessive pressure resulted in greater expiratory WOB and as a consequence a ~33% greater V˙  O2RM for that trial.  2.4  Discussion Overview. We have presented methodology allowing for the accurate replication of exercise hyperpnea at rest and the subsequent calculation of V˙  O2RM .  During exercise, breathing strategy is optimized in order to provide sufficient V˙  E with minimal effort (102).  As such, to accurately estimate the V˙  O2RM incurred during exercise, all ventilatory parameters must be carefully considered and replicated.  As V˙  O2RM is linearly related to work, the most crucial variable to replicate when estimating V˙  O2RM is the WOB.  Specifically, the WOB needs to be directly assessed or potentially large errors could go unnoticed (for example, see Figure 5).  We used customized software that allowed for the on-line visualization of pressure-volume and flow-volume loops overlaid on a fixed target.  The novel implementation of dual visual feedback of flow and pressure allowed us to consistently replicate exercise WOB during the mimicking trials.  With the added feedback, our ability to accurately and consistently replicate exercise parameters and WOB (Figure 2 and Table 2) was improved compared to other investigations.  Specifically, our WOB CV was smaller (16.2 vs. 11.1%) resulting in a decreased V˙  O2RM  23  CV (10.4 vs. 5.7%) when compared to others (2).  Overall, given sufficient familiarization, trial length, number of trials and extensive feedback the estimation of exercise V˙  O2RM is possible and consistent.  Familiarization day. The familiarization day improved matching of VT and V˙  E, whereas, fR was not statistically improved.  Breathing frequency was continuously paced using a metronome and all subjects were immediately able to match their target consistently; thus, we did not anticipate any learning or practice improvement.  Conversely, feedback of VT and V˙  E was updated every breath and no auditory cue was used.  After several practice trials, subjects were better able to pace the force of their breath so they inspired/expired the correct volume at the fR auditory cues.  A common problem was subjects would inspire the intended volume faster than their target TI, the result of higher initial flows.  In these trials, after the target was reached, there would be a pre-expiratory pause or only minor inspiratory flow for the latter part of inspiration, resulting in an unnatural breathing pattern.  This pattern would not be obvious if only VT, fR and V˙  E were assessed, but was readily apparent when viewing on-line flow-volume loops.  As such, we provided cueing and feedback to subjects to identify and correct the patterns during familiarization trials.         While the gains in accuracy were relatively small (~5%), we believe the familiarization day was important and necessary given the sensitivity of estimating V˙  O-2RM .  The familiarization day also gave subjects experience matching their flow-volume loops in real time.  Thus, on the experimental days, they would be able to concentrate on accurate replication of pressure-volume loops and not be overwhelmed by the extensive feedback.   24  Improvements. The variable that we, and others (2), had the most difficulty replicating was EELV.  As seen in Figure 2, at higher lung volumes there was a bias towards performing the hyperpnea at an increased EELV.  We were only able to provide subject feedback with a surrogate measure (end-expiratory esophageal pressure), as assessment of EELV necessitates altering breathing pattern (IC maneuver) or inspired gases (inert gas dilution).  Two methods of obtaining continuous lung volume would be utilizing opto-electronic plethysmography or respitrace-bands.  Using opto-electronic plethysmography, one could determine the continuous change in volume in the abdomen and thorax (20) and quantify any configurational changes.  While respitrace-bands could provide estimates of volume changes around two compartments.  Despite the bias, we were still able to effectively match the WOB during those trials; therefore we do not expect this overestimation to affect our results.  Our subjects mimicked their breathing pattern for 5 minutes, which was longer than each respective exercise stage they were replicating (2 minutes).  At higher V˙  E (above 80-85% V˙  Emax) could this duration of hyperpnea result in fatigue of the respiratory musculature?  Evidence suggests that in the absence of other exercise related stimuli (pH, heat, metabolites) sustained exercise hyperpnea is not fatiguing per se (3, 16).  The critical variable related to fatigue development appears to be WOB, as its manipulation will determine the presence or absence of diaphragm fatigue (15).  We took several steps to prevent diaphragm fatigue.  First, we monitored heart rate and blood pressure between trials to ensure the rest period (~10+ minutes) was of sufficient length that they returned to baseline.  Second, the stages were randomized with appropriate rest between stages and no effect of order was observed.  Third, the mimicking trials were completed over two days.  Fourth, each trial was limited to 5 minutes in length, which is significantly less time than when task failure normally occurs  (3).  Fifth, we controlled the WOB so that it was similar to exercise.  Ensuring that the WOB was not excessive was the most  25  important control measure of this study, as resting hyperpnea with substantially higher WOB has been shown to be fatiguing (16).  Based on the above we contend that our subjects did not develop fatigue.  On the other hand, we did not specifically test for respiratory muscle fatigue, nor do we know the effects, if any, it would necessarily have on V˙  O2RM .  A final caveat is that over the course of an incremental exercise test there is an increase in core temperature along with marked bronchodilation.  In the present study, we did not control for these factors but acknowledge they could have influenced metabolism and respiratory mechanics during our mimic trials.  Work of breathing. An accurate replication of the exercise WOB is the most critical aspect when estimating the V˙  O2RM during voluntary hyperpnea.  During exercise, breathing mechanics are regulated to ensure the most efficient pattern is adopted.  It has been demonstrated that transpulmonary pressure can meet, but rarely exceeds the threshold where gains in flow no longer occur (75).  The latter example of optimizing driving pressure is illustrated in Figure 5.  In this example, the subject was mimicking their V˙  Emax, where they developed EFL during exercise and when mimicking the same V˙  E.  In one trial, the subject was expiring more forcefully, as indicated by the greater esophageal pressure on expiration (Panel B).  The added pressure is “wasted” because it cannot alter the driving gradient for flow and only increases the WOB (+80 J min-1).  Additionally, the excessive pressure could further compress the airways, increasing the resistive component of the already greater total WOB.  In our example (Figure 5), all of the increase in WOB was due to resistive factors and the expiratory portion of the WOB was more than double in the excessive trial.  The use of on-line feedback of pressure-volume loops minimized this potentially critical error.  By having the subjects replicate exercise pressures, we are confident that any EFL or airway compression was similar during the hyperpnea trials.  The idea of EFL representing a continuum (or “impending  26  EFL”) has been suggested previously (75) but has now received more widespread attention (12, 45).  Specifically, there are likely some reflexive or mechanical changes when approaching the effective driving pressure.  As such, to accurately recreate this phenomenon during resting hyperpnea, the dynamic changes in pressure throughout a breath need to be assessed, which is possible given our on-line feedback.   Figure 5. Example of inaccurately high V˙  O2RM  values due to excessive work of breathing. Both trials are from the same male subject mimicking their maximal exercise breathing pattern.  The dotted line represents a trial in which excessive pressure was generated,  27  whereas the solid thin lines represent a trial in which they accurately matched their exercise work of breathing.  Grey stippled area indicates the excessive pressure generated (panel B) and the corresponding change in flow (panel A)  End-tidal carbon dioxide. Unique to our study was the precise replication of exercise  values during the hyperpnea trials.  Commonly, iso-capnia is maintained by using pre-mixed inspired gases with FICO2 ranging from 4-6% and similar fractions are given to all subjects.  However, this method is not optimal as evideent by the large inter-subject variability in exercise PETCO2 .  The concern is that along with marked differences in V˙  Emax (range 91-191 l min-1) individual relative end-exercise hyperventilatory responses are different  resulting in varying end-exercise PETCO2 (33).  Therefore, during replication of maximal exercise hyperpnea we used a FICO2 between 2.2-4.0%.  As arterial CO2 tensions are potent vasoactive stimuli (26), not replicating exercise values could result in altered respiratory muscle (or other vascular bed) perfusion.  In the current study, we erred on the side of lower PETCO2 during mimicking when values were above rest during exercise (Figure 2, panel D).  We chose this approach because PETCO2 during exercise remains near rest and will decrease later in exercise (Dominelli et al., 2013).  As such, the relative mild hypocapnia represents a physiologically relevant state in healthy individuals during exercise, therefore we do not expect it to have altered our results.  Number and duration of trials. Our subjects performed multiple trials of each stage to generate a V˙  O2RM estimate.  Even when replicating all the parameters precisely, there was some variation V˙  O2RM (Table 2) due to factors that are uncontrollable such as slight variations in substrate utilization, non-respiratory muscle activation and increased cardiac oxygen consumption.  We chose to have subjects perform several trials in succession and report these as an average, in essence reporting the V˙  O2RM for a given V˙  E as a range rather than a specific value.  28   For each of the hyperpnea trials, we had subjects replicate their breathing pattern for 5 minutes and we averaged the last minute to calculate V˙  O2RM.  The length of the trial was desired for several reasons.  First, there is an un-physiological transient rise and fall in V˙  O2 for ~1 minute starting at the beginning of each trial (Figure 3, panel A).  This lag in equilibrium between mixed expired and alveolar gases results from metabolic and/or circulatory delays.  Extrinsic factors responsible for the lag include the pump speed and response time of the gas analyzers and size of the mixing chamber.  Intrinsic factors such as systemic blood transit time, heart rate and muscle perfusion could also affect the lag time.  The remaining period (~4 minutes) allowed us to coach the subject into the most accurate replication of their exercise-breathing pattern and reach steady state.  The end result was that mixed expired gases showed little variation throughout the final aspect of the trial, where the average was taken.  The importance of stable mixed expired gases is appreciated when we considered the variables for calculating V˙  O2, namely the true oxygen difference (difference between FIO2 and FEO2 after accounting for water vapor) and V˙  E (135).  During various intensities of exercise, the true oxygen difference is ~3-5%, which is then multiplied by V˙  E (after appropriate environmental corrections).  Conversely, during the resting hyperpnea trials, the true oxygen difference was ~0.5%.  Thus, while the V˙  E is similar between both conditions, the other variable when calculating V˙  O2 (true oxygen difference) is markedly smaller.  As such, a similar 0.1% measurement error in gas concentration would result in an approximately ten times greater error in V˙  O2 during voluntary hyperpnea compared to exercise.  In our view, this relationship stresses the importance of accurate and consistent measurement of expired and inspired gases.  Recommendations and future direction for estimating V˙  O2.  We have demonstrated that the V˙  O2RM incurred during exercise can be reliably estimated, however, care and attention to specific details are required.  Replicating exercise hyperpnea at rest is the most  29  physiologically relevant method for V˙  O2RM assessment because it represents a natural breathing pattern.  Our specific recommendations are as follows.  First, during hyperpnea trials, the exercise WOB must be precisely replicated, which requires utilizing an esophageal balloon catheter.  It is insufficient to merely match end-inspiratory and end-expiratory pressure; rather, a system to match the dynamic pressure changes throughout a breath (see figure 5) is needed.  Second, PETCO2 during a hyperpnea trial should be identical to those experienced during the mimicked exercise for each subject.  Third, trials must be of sufficient length in order for steady state to occur, which will depend on experimental set-up and subject’s ability to match exercise variables.  We found that 5-minutes trials were adequate without being excessively long.  Fourth, each stage of exercise should be mimicked multiple times as there is inherent variability even when breathing patterns are controlled (Table 2).  Finally, before any experimental trials are conducted, the subjects should be familiarized with the set-up and feedback they will receive during the hyperpnea.    While there was no difference between men and women in their ability to mimic their exercise hyperpnea (Table 2), could there be an effect of sex on the V˙  O2RM/ V˙  E relationship?  The current study was not designed or intended to study the effect of sex on V˙  O2RM, but given the many sex difference in the respiratory system (Sheel et al., 2004) the hypothesis is justified.  To determine if there is a sex difference in the V˙  O2RM a larger sample size is required and participants should replicate a broad range of exercise V˙  E.  Finally, the additional effect of mechanical ventilatory constraints would also require consideration.       Conclusion. In conclusion, the accurate replication of all respiratory parameters is required for appropriate estimations of V˙  O2RM.  Specifically: subjects should be familiarized with the experimental set-up, a sufficient number of trials at each target V˙  E  30  need to be performed, each trial needs to be of adequate length to ensure steady-state V˙  O2 all variables associated with the exercise V˙  E need to be mimicked and the WOB needs to be identical to that of exercise.  31   Chapter Three: Oxygen cost of exercise hyperpnoea is greater in women compared with men  3.1 Introduction During dynamic exercise, ventilation rises in proportion to the metabolic demands of the locomotor muscles.  This exercise-induced increase in minute ventilation (V˙  E) results in an increased mechanical work of breathing (WOB) (102).  Consequently, the metabolic and circulatory costs of exercise hyperpnoea are substantial.  In healthy untrained humans, the oxygen uptake of the respiratory muscles (V˙  O2RM) during maximal exercise represents ~10% of whole-body maximum oxygen uptake (V˙  O2max) (3, 97, 121).  In endurance trained men, who possess a high V˙  O2max and sustain high rates of ventilation, the V˙  O2RM can represent upwards of 15% of V˙  O2max (3).  Akin to other skeletal muscles, the contracting respiratory musculature requires sufficient blood flow to meet oxygen demand (8).  Whilst direct blood flow measurements are not available in humans, at maximal exercise in ponies ~15% of cardiac output is directed towards the respiratory muscles (85); this value is commensurate with estimates of blood flow in humans (23, 56).  Quantifying the metabolic demands of the respiratory muscles is necessary for understanding cardiorespiratory control during exercise.  Specifically, high respiratory muscle work has been shown to alter blood flow distribution (54) and reduce cardiac output by altering preload and afterload (56).  Similar alterations in cardiac output have also been demonstrated with externally imposed expiratory loading (95, 125).  There are well-documented sex differences with respect to airway anatomy and the respiratory mechanics associated with exercise hyperpnoea.  When compared to height matched men, women have smaller lungs (127) and conducting airways (90, 120).  During dynamic whole-body exercise, women develop expiratory flow limitation (EFL) more often (53, 87) and have a greater mechanical WOB for a given V˙  E above ~65 l min-1  32  (51, 134).  It has been hypothesised that mechanical ventilatory constraints, such as EFL, are associated with a higher V˙  O2RM (3), although to our knowledge this has not been systematically investigated.  Therefore, it is important to consider the potential independent effect of EFL on V˙  O2RM.  There have been limited attempts to compare the V˙  O2RM between men and women (43, 81, 131).  Unfortunately, methodological inadequacies and conflicting results render the findings difficult to interpret.  For example, most of the studies assessed the V˙  O2RM in men and women at ventilations where sex-difference may not necessarily be present (<60 l min-1).  The importance of determining sex-based differences in V˙  O2RM is because cardiac output is finite during maximal exercise.  As such, if women have a greater V˙  O2RM and must therefore dedicate a greater fraction of total blood flow towards their respiratory muscles during maximal exercise, performance may be impaired due to reduced locomotor muscle blood flow (57). Accordingly, we sought to compare V˙  O2RM in men versus women to address whether, for a given ventilation, women have a greater absolute V˙  O2RM and whether this represents a larger percentage of whole-body V˙  O2 compared with men.  A secondary aim was to determine the effect of EFL on V˙  O2RM.  We hypothesized that at submaximal and maximal ventilations where the WOB is greater compared to men, women have a greater V˙  O2RM and this constitutes a larger proportion of whole-body oxygen uptake.  We further hypothesized that at maximal exercise, those who develop EFL will have a higher V˙  O2RM.  33  3.2 Materials and methods Subjects.  After providing written informed consent, eighteen (9 male, 9 female) healthy subjects participated in the study.  Some subjects (13 of 18) had previously participated in a study designed to determine the reproducibility of V˙  O2RM (39).  The primary outcome measures in the current study did not overlap with any of the previous analyses.  All procedures adhered to the Declaration of Helsinki and were approved by the Clinical Research Ethics Board at the University of British Columbia.  Subjects had a wide range of exercise participation (recreational to national calibre athletics), did not report any current or previous cardiorespiratory ailments and had spirometry within normal limits (127) (Table 3).  Although not universally established, studies that have measured conjugates of oestrogen and progesterone have demonstrated significant inter- and intra-subject variability with respect to hormone concentrations throughout the menstrual cycle, but with no effect on submaximal exercise ventilation (19, 84).  Therefore, we tested female subjects at random points throughout their menstrual cycle and oral contraceptive use was not an exclusion criterion.  Table 3. Anthropometric and spirometric values.  Men (n=9) Women (n=9) Age (y) 29±3 23±1* Height (cm) 183±2 167±2* Mass (kg) 75±3 58±2* FVC (l) 5.8±0.2 4.0±0.2* FVC (% predicted) 99±4 95±2 FEV1 (l) 4.7±0.2 3.4±0.1* FEV1 (% predicted) 100±4 94±2 FEV1/FVC 82±2 85±1 FEV1/FVC (% predicted) 100±2 99±2 FVC, forced vital capacity; FEV1, forced expired volume in 1 second; PEF, peak expiratory flow. * Significantly different compared to men (P<0.05).  Values are mean±SE.   34   Experimental design.  Subjects completed 4 days of testing.  Day 1 consisted of maximal incremental cycle exercise in order to obtain spontaneous ventilatory parameters during exercise.  Day 2 served to familiarize subjects with the voluntary hyperpnoea protocol used to estimate V˙  O2RM.  Days 3 and 4 were experimental days, during which subjects mimicked their exercise breathing patterns at rest while V˙  O2RM was assessed.  Days 1 and 2 were separated by at least 48 h, whereas the experimental days were separated by at least 24 h.  Subjects were instrumented with oesophageal and gastric catheters on days 1, 3 and 4 for the assessment of respiratory pressures. Maximal exercise (day 1).  To obtain spontaneous breathing patterns, a step-wise incremental test on a cycle ergometer (Excalibur Sport; Lode, Groningen, The Netherlands) was performed to the limit of tolerance after insertion and placement of oesophageal and gastric balloon-tipped catheters.  To ensure subjects exercised for similar durations, men began at 120 W and women began at 80 W, with a 20 W increase every 2 min for both groups.  Testing was terminated when subjects could not maintain >60 rpm despite encouragement.  Cardiorespiratory variables including EFL were assessed using customized hardware and software as described elsewhere (35, 39). Table 4. Cardiorespiratory values at maximal exercise.  Men (n=9) Women (n=9) V˙  O2 (l min-1) 4.4±0.2 2.8±0.2* V˙  O2 (ml kg-1 min-1) 58.7±1.9 48.1±2.1* Range 50.3-68.5 41.4-60.4 V˙  CO2 (l min-1) 4.8±0.2 3.0±0.1* VT (l) 3.1±0.1 1.9±0.1* fb (breaths min-1) 56±3 61±3 V˙  E (l min-1) 173±10 114±4* RER 1.11±0.02 1.10±0.02 HR (beats min-1) 183±2 189±3 PETCO2 (mmHg) 28±1 28±1 V˙  E/ V˙  CO2 36±1 38±1 V˙  E/ V˙  O2 40±2 42±2 EELV (% FVC) 40±2 43±2 EILV (% FVC) 88±1 87±1 ΔPoe (cmH2O) 54±4 46±1*  35   Men (n=9) Women (n=9) WOB  (J min-1) 605±59 354±19* PTPoe (cmH2O s-1 min-1) 606±35 500±30* PTPdi (cmH2O s-1 min-1) 457±44 406±75 PTPoe/PTPdi 0.77±0.07 0.84±0.11 V˙  ECap (l min-1) 220±15 164±9* V˙  E/ V˙  ECap (%) 80±3 72±5 EFL (%) 23±9 21±8 EFL (n) 5 5  V˙  O2, oxygen uptake; V˙  CO2, carbon dioxide output; VT, tidal volume; fb, breathing frequency; V˙  E, expired minute ventilation; RER, respiratory exchange ratio; HR. heart rate; ΔPoe, oesophageal pressure swing; PETCO2, end-tidal carbon dioxide tension; EELV, end-expiratory lung volume; EILV, end-inspiratory lung volume; WOB, work of breathing; PTPeso, oesophageal pressure-time product; PTPdia, diaphragmatic pressure-time product; V˙  ECap, ventilatory capacity; EFL, expiratory flow limitation. * Significantly different compared to men (P<0.05).  Values are mean±SE.  Voluntary hyperpnoea (days 3 and 4).  To estimate V˙  O2RM, subjects rested on the cycle ergometer and mimicked tidal volume, frequency, V˙  E, duty cycle and respiratory pressures associated with their exercise hyperpnoea (39).  End-tidal CO2 tension was set to a level similar to each respective exercise stage.  Briefly, each subject mimicked the breathing pattern associated with 4-5 submaximal exercise stages and maximal exercise.   The experimental trials were performed in random order over two days.  Each stage was mimicked 4-6 times sequentially and each trial was 5 min in duration.  The first 4 min was used to provide feedback and ensure sufficient time for mixed expired gas fractions to reach a steady-state.  The final minute of each stage was used for subsequent analysis.  During each of the voluntary hyperpnoea trials, the subjects maintained the same body position as during cycle exercise.  Ample rest was allowed between trials, ranging from 5 min for lower intensity V˙  E (40% V˙  Emax) to >30 min for higher V˙  E (>75% V˙  Emax).  Heart rate and blood pressure were measured during the rest periods to ensure complete recovery before the subsequent hyperpnoea trial.  Similar clothing was worn during exercise and voluntary hyperpnoea to avoid any clothing-induced changes in pulmonary mechanics and the ergometer configuration was identical for both conditions.  We  36  included a familiarization day as it significantly improves the accuracy of tidal volume and V˙  E and the matching of expiratory flow during voluntary hyperpnoea (39).  During the familiarization day, each hyperpnoea trial was mimicked several times and similar feedback (except oesophageal and gastric pressure) was provided.    Figure 6. Oesophageal pressure-volume loops for a representative male subject during exercise (Pane A-D) and voluntary hyperpnoea (Panel E-H). Loops representing exercise are an average composite from 30 seconds of data.  Loops representing voluntary hyperpnoea are average composites of the final minute of several trials.  Ventilation and work of breathing are presented in tables for each respective stage.  V˙  Emax, maximum minute ventilation; Ex, exercise; Hyp; hyperpnoea; V˙  E, expired minute ventilation; WOB, work of breathing.   37  Several procedures were used to ensure that the ventilatory responses to exercise were replicated during the voluntary hyperpnoea trials (39).  Numerical tidal volume was displayed to the subjects on a breath-by-breath basis and verbal feedback was provided throughout.  Breathing frequency and duty cycle were maintained by breathing in time to a metronome.  Directly in front of the subjects was a screen displaying online flow-volume and pressure-volume loops that were overlaid on loops obtained during exercise.  Representative traces of a subject’s oesophageal pressure during exercise and voluntary hyperpnoea are shown in Figure 6.  Subjects were coached by the same investigator to match their on-line flow-volume and pressure-volume loops with the target loop.  End-expiratory lung volume was monitored using the surrogate measure of end-expiratory oesophageal pressure.  During each trial, the inspired gas was modified to ensure end-tidal CO2 tension was similar to the respective exercise stage.  In this regard, inspired percentage of CO2 ranged from 2-6% whereas inspired O2 was always ~21%.           In total, 442 experimental trials were completed, with equal distribution between the sexes.  Each subject performed 20-30 trials, equally distributed across the experimental days.  To determine the between-day reproducibly of V˙  O2RM, one subject repeated the entire procedure six months after initial testing; all estimates were within 5-7% of the original measures over their full range of V˙  E (40-150 l min-1).  Data analysis.  Data were collected using a 16 channel analogue-to-digital data acquisition system (PowerLab/16Sp model ML 795, ADInstruments, Colorado Springs, CO, USA), sampled at 200 Hz, and stored on a computer for subsequent analysis using bespoke software (GNARx, developed in LabView 2013, Austin, Tx, USA). The final 30 s of each exercise stage was used for subsequent analysis.  During voluntary hyperpnoea, the final minute was used for analysis.  A longer time was used for the voluntary hyperpnoea protocols to ensure steady-state V˙  O2.  Flow-volume and pressure-volume loops for each trial were constructed from ensemble-averaged breaths.  Tidal flow- 38  volume loops were placed within the maximal expiratory flow-volume (MEFV) envelope by determining end-expiratory lung volume from an inspiratory capacity manoeuver (50).  The WOB, extent of EFL and ventilatory capacity were determined using previously described methods (33).  Efficiency of the respiratory muscles was estimated by dividing the measured V˙  O2RM by the ideal oxygen uptake needed to perform the WOB (2).  To calculate the ideal oxygen uptake, the measured WOB was converted into units of oxygen with changes in respiratory exchange ratio accounted for.  The maximal effective ventilation was defined as when the change in V˙  O2RM per unit of V˙  E equalled the change in whole body V˙  O2 per unit V˙  E (101, 121).  Once the maximal effective ventilation was determined, maximal exercise gas exchange parameters and the alveolar gas equation were used to predict the maximal alveolar ventilation and the corresponding V˙  O2.   Statistics.  Anthropometric and maximal exercise variables were compared between men and women using independent samples t-tests.  An equation was fitted to each subject’s relationship for WOB vs. V˙  E, V˙  O2RM vs. WOB and V˙  O2RM vs. V˙  E.  The V˙  O-2RM vs. WOB relationship was fit with a linear equation, whereas the WOB vs. V˙  E and V˙  O2RM vs. V˙  E were fit with an exponential equation.  The respective constants for each equation were pooled and the sexes were compared using independent samples t-tests.  To determine the specific V˙  E or WOB for which the groups were different, each subject’s equation was solved for successive independent variables, with the resultants compared with t-tests and Bonferroni correction.  To determine sex-independent differences, subjects were grouped into those with and without EFL during exercise and similar comparisons were completed.  Expiratory flow limitation was defined as >5% overlap of the tidal flow-volume loop with the MEFV curve.  The effect of sex and EFL was also compared at different percentages of V˙  Emax (45, 60, 75 and 100%) using a two-factor (sex and % V˙  Emax) repeated-measures ANOVA.  When significant F ratios were detected, Tukey’s post-hoc test was conducted.  The percentages of V˙  Emax were selected because:  39  (i) they spanned a wide range of ventilation, (ii) they represented ~10% increments in V˙  O2max (see Table 6) and (iii) all subjects had mimicked an exercise stage within this range.  Statistical significance was set at P<0.05.  All values are presented as mean ± SE unless otherwise noted. 40  3.3 Results Subject characteristics and cardiorespiratory responses. Subject characteristics are presented in Table 4.  Maximal cardiorespiratory and respiratory mechanics values are presented in Table 5.  At peak exercise, men had significantly greater absolute and relative oxygen uptake, carbon dioxide output, tidal volume and V˙  E (P<0.05).  However, there were no differences in respiratory exchange ratio, heart rate, end-tidal CO2, ventilatory equivalents or operational lung volumes (P>0.05).  At maximal exercise, men had significantly greater WOB owing to a greater V˙  E (P<0.05).  For a given V˙  E, however, women had a greater WOB due to a significantly greater resistive component (P<0.05) (Figure 7 and 8).  Figure 7. Relationship between work of breathing and minute ventilation during the voluntary hyperpnoea The work of breathing is significantly greater in women at and above a ventilation of ~75 l min-1.  WOB, work of breathing; V˙  E, expired minute ventilation.   41  Work of breathing and V˙  O2RM.  Figure 9 shows the absolute and relative V˙  O2RM at different absolute and relative ventilations.  While there was minimal within-subject variability for V˙  O2RM, there was greater between-subject variability which was more pronounced at higher ventilations (Panel A).  At a V˙  E of ~95 l min-1, for example, the V˙  O-2RM in women ranged from 200-400 ml min-1.  The variability in V˙  O2RM is explained by differences in the WOB vs.  V˙  E relationship, which is dependent on airway size and presumably different in our groups.  The order of trials had no effect on V˙  O2RM.  Specifically, when replicating maximal exercise ventilation the average V˙  O2RM for all subjects was not statistically different across trials (381 ± 32, 377 ± 31, 404 ± 34 and 389 ± 31 ml O2 min-1 for the 1st-4th trials, respectively; P>0.05).  There was also no effect of order when the subjects were grouped by sex (P>0.05).  At an iso-ventilation of ~55 l min-1, women had a greater absolute V˙  O2RM (Panel B) (P<0.05).  The group mean coefficient of variation for V˙  O2 was 4.9, 4.9, 5.3 and 6.0% at 45, 60, 75 and 100% V˙  Emax, respectively, with no difference between the sexes (P>0.05).  When compared at relative ventilations, men and women had a similar absolute V˙  O2RM (P>0.05, Panel C).  However, this represented a greater fraction of whole-body V˙  O2 in women at 75 and 100% V˙  Emax (Panel D).  At maximal exercise, V˙  O2RM represented 13.8% of whole body V˙  O2 in women and 9.4% in men (P<0.05, Figure 10).    42   Figure 8. Relationship between elastic (Panel A) or resistive (Panel B) work of breathing and minute ventilation Asterisk represents a greater resistive work of breathing in women at iso-ventilation (P<0.05).  Dagger represents a significantly greater elastic work of breathing and resistive work of breathing in men when maximum exercise ventilations are compared (P<0.05).  V˙  E, expired minute ventilation.   Without exception, every subject demonstrated a significant linear relationship between V˙  O2RM and WOB.  During maximal intensity trials, however, the V˙  O2RM rose out of proportion to the increase in WOB (Figure 11).  When WOB was related to V˙  O2RM as a % of total whole-body V˙  O2, the average slope of the regression line was significantly greater in women (P<0.05, Figure 11B).   43   Figure 9. Panel A: average oxygen uptake for each stage of voluntary hyperpnoea performed by each subject.  Panel B: regression lines for men and women performing voluntary hyperpnoea trials.  The star, vertical line and arrow indicate that women have a significantly higher V˙  O2RM above a ventilation of ~55 l min-1.  Regression was fitted using the average of each subject’s constants.  Panel C: absolute V˙  O2RM at different percentages of maximal ventilation.  Men had significantly greater ventilations at every comparison (see also Table 3).  Panel D: V˙  O2RM as a percentage of whole-body oxygen uptake at different percentages of maximal ventilation. All average values are mean ± SE.  V˙  E, expired minute ventilation; V˙  Emax, maximum minute ventilation; V˙  O2, oxygen uptake; V˙  O2max, maximum oxygen uptake; V˙  O2RM, oxygen uptake of the respiratory muscles. * Significantly greater in women (P<0.05).   Table 3 displays variables for men and women during the voluntary hyperpnoea trials at different %V˙  Emax.  Men had greater absolute V˙  E for all comparisons (P<0.05), primarily due to greater tidal volume.  The changes from rest to hyperpnoea in heart rate, expiratory duty cycle and end-tidal carbon dioxide tension were not different between the  44  sexes at any %V˙  Emax (P>0.05).  The V˙  O2RM/V˙  E relationship increased in both sexes as V˙  E increased, and men were statistically lower at 75 and 100% V˙  Emax (P<0.05).  The calculated efficiency of the respiratory muscles was greater in men at all ventilations (P<0.05).  Women performed the hyperpnoea trials at significantly higher end-expiratory lung volume (P<0.05), with no difference in end-inspiratory lung volume (P>0.05).  Expiratory flow limitation.  The effect of stratifying the subjects based on the appearance of EFL on V˙  O2RM is shown in Figure 12 and Table 7.  There were similar numbers of men and women in each group and there was no difference between %EFL during exercise or hyperpnoea for either sex (men: 24±9% vs. 23±9%; women 22±8% vs. 18±6%; for exercise and hyperpnoea, respectively; P>0.05 for both).  The MEFV curve was significantly larger in the group that did not develop EFL (Panel C, P<0.05).  There were no differences in V˙  O2max or V˙  Emax (P<0.05); however, the V˙  O2RM was significantly greater in the EFL group during the maximal ventilation trials (P<0.05, Panel B).  At ≤75% V˙  Emax, where there was no or minimal EFL (3 subjects, <20% overlap with the MEFV curve), the V˙  O2RM and V˙  E were similar between groups (Panel B).  Although the absolute WOB was not different between groups (485±63 vs. 443±63 J min-1, P>0.05), the resistive work contributed a significantly greater percent in the EFL group (79±1 vs. 72±2% of total WOB, P<0.05).  At all ventilations lower than 100%, there were no differences between the EFL and NEFL groups (Table 4).  At 100% V˙  E, the EFL group had a greater breathing frequency and V˙  O2RM per V˙  E and a lower estimated respiratory muscle efficiency (P<0.05, Table 7).  45  3.4 Discussion Major findings.  The major findings from this study are three-fold.  First, women have a greater absolute V˙  O2RM during submaximal and maximal rates of exercise hyperpnoea.  Second, during strenuous and maximal exercise, the V˙  O2RM represents a significantly greater fraction of whole-body oxygen uptake in women.  Finally, regardless of sex, those who develop EFL at maximal exercise have a greater V˙  O2RM.  Collectively, our findings indicate that the greater WOB and increased mechanical ventilatory constraints in women result in a greater absolute and relative V˙  O2RM at submaximal and maximal exercise intensities.   Figure 10. Box and whisker plot, showing individual subject data and group mean ± SE for V˙  O2RM as a percentage of whole-body oxygen uptake at maximal exercise in women and men. Squares in the box and whisker plot represent 5th and 95th percentiles and the horizontal line is the median.  V˙  O2RM, oxygen uptake of the respiratory muscles; V˙  O2max, maximum oxygen uptake. * Significantly greater compared with men (P<0.05).   46   Sex-differences in V˙  O2RM.  We demonstrated that women have a greater absolute and relative V˙  O2RM compared to men.  Given that oxygen uptake is linearly related to work, we hypothesized that at ventilations where women have a greater WOB than men, their V˙  O2RM would also be greater.  Indeed, we found that women had a greater WOB at submaximal V˙  E (Figure 12), a finding consistent with work from our laboratory (53) and others (134).  The differences in WOB were due to increased resistive work (Figure 8).  We found differences in V˙  O2RM between the sexes at ~55 l min-1, which coincides with the V˙  E where the WOB is greater in women.  A ventilation of ~55 l min-1 was achieved during submaximal exercise in both men and women, but represented a greater fraction of maximal ventilation in women.  Therefore, we also used relative units to compare the sexes at similar fractions of V˙  Emax.  As shown in Figure 9C, when compared at similar relative V˙  Emax, there were no differences in the absolute V˙  O2RM.  However, for the comparisons presented in Figure 9C, the men had a significantly greater V˙  E and their whole-body V˙  O2 was also greater (Table 6).  To fully elucidate the comparisons, units on each axis should be relative, as shown in Figure 4D which displays the comparison between relative V˙  E and relative   V ˙ O2RM.  At ≥75% V˙  Emax (~90% of V˙  O2max), a significantly greater fraction of whole-body V˙  O2 was dedicated to the respiratory muscles in women.  If the respiratory muscles in women command a greater percent of cardiac output, then blood flow to locomotor muscles may become compromised (see Perspectives)  47   Figure 11. Oxygen uptake of the respiratory muscles in absolute (Panel A) and relative units (Panel B) at different WOB for each subject performing each trial. Group regression lines were developed by averaging each subject’s regression and producing a composite.  All subjects demonstrated a significant relationship between V˙  O-2RM and WOB (P<0.01).  For Panel A, there was no difference in the intercepts of the regression lines, but women had a significantly greater slope (1.29 ± 0.12 vs. 0.85 ± 0.05, P<0.05).  Similarly, for Panel B, there was no difference in the intercepts of the lines, but women had a significantly greater slope (0.034 ± 0.002 vs. 0.012 ± 0.001, P<0.05).   V˙  O-2RM, oxygen uptake of the respiratory muscles; WOB, work of breathing.  Both sexes showed an increase in the unit rate of V˙  O2RM per V˙  E and were not different at 45 and 60% of V˙  Emax.  The progressive increase in V˙  O2RM/ V˙  E as ventilation increased has been observed by others (3, 24).  At 75% of V˙  Emax, however, women showed marked increase in V˙  O2RM/ V˙  E, whereas men only demonstrated a dramatic increase between 75 and 100% V˙  Emax (Table 3).  While the V˙  O2RM/ V˙  E increased with ventilation, the V˙  O2RM/WOB relationship did not change systematically in either sex and was consistently greater in women.   Despite a linear relationship for V˙  O2RM/WOB in both sexes, the efficiency of the respiratory muscles was significantly lower in women and this finding was most pronounced at maximal exercise (Table 6).  While our study  48  was not designed to determine the mechanism behind sex-based differences in respiratory muscle efficiency, our observations merit brief comment.  Our primary concern with determining efficiency is that we are unable to account for all of the work done during breathing.  For example, abdominal muscles contract to stabilize the abdominal wall during forceful expiration  (27), and work is done when the chest-wall is distorted at near maximal ventilations (>75% of V˙  Emax) (49).  Indeed, the work done to stabilize the abdominal wall and distort the chest wall is estimated to be upwards of 25% of total WOB (47).  A further consideration is that the velocity of muscle shortening would have been greater in the women due to a higher maximal breathing frequency (Table 6), and this could also have necessitated greater work in order to stabilize the abdominal wall.  Furthermore, the greater shortening velocity in women would be expected to result in an increased energy requirement (88, 89).  Accordingly, we cautiously speculate that decreased respiratory muscle efficiency in women could arise from sex-differences in substrate utilization and morphology (60, 94) and/or blood vessel compression when the WOB is near maximal (67).   49  Table 5. Cardiorespiratory variables during voluntary hyperpnoea at different percentages of maximal exercise ventilation.  45% V˙  Emax 60% V˙  Emax 75% V˙  Emax 100% V˙  Emax  Men Women Men Women Men Women Men Women V˙  O2max (%max) 70±3 72±3 83±1 83±2 92±1 92±2 100 100 VT (l) 3.1±0.2 1.7±0.1* 3.3±0.2 1.8±0.1* 3.3±0.2 1.9±0.1* 3.1±0.2 1.8±0.1* fb (breaths min-1) 27±2 34±2* 33±2 39±2 41±3 46±2 53±2 60±2* ΔHR (beats min-1) 0±3 -2±3 7±2 6±4 7±1 11±3 20±3 25±3 Te/Ttot  0.55±0.01 0.55±0.01 0.54±0.01 0.55±0.01 0.53±0.01 0.55±0.01 0.51±0.01 0.53±0.01 PetCO2 (mmHg) 40±1 37±1 40±1 37±1 35±1 36±1 31±2 35±2 V˙  E (l min-1) 81±7 55±2* 106±5 71±3* 133±7 88±3* 165±4 109±4* V˙  O2RM/ V˙  E (ml O2 l-1) 1.4±0.1 1.5±0.1 1.7±0.1 1.9±0.1 1.7±0.1 2.4±0.2* 2.4±0.2 3.5±0.3* V˙  O2RM/WOB (ml O2 J-1) 0.8±0.1 1.1±0.1* 0.8±0.05 1.2±0.1* 0.7±0.04 1.1±0.1* 0.7±0.0 1.1±0.1* EfficiencyRM (%)  6.7±0.5 5.1±0.6* 6.4±0.4 4.4±0.3* 7.7±0.94 4.8±0.3* 7.2±0.2 4.6±0.4* ΔPoe (cmH2O) 24±1 19±2 31±2 24±2 39±3 32±2 55±4 47±3* WOB (J min-1) 142±21 75±8 232±23 122±12* 347±38 199±15* 593±60 339±27* WOBres (% total) 40±3 51±5* 57±3 54±5 65±3 65±4 76±2 76±3 WOBel (% total) 60±3 49±6* 43±3 46±5 35±3 35±4 24±2 24±3 EELV (% FVC) 35±2 44±3* 33±1 44±3* 32±1 43±2* 38±2 46±3* EILV (% FVC) 83±2 83±3 85±2 86±3 85±2 88±2 87±1 88±1 V˙  E/ V˙  ECap (%) 38±3 33±3 52±3 40±3* 67±3 51±3* 74±4 67±4  V˙  E, minute ventilation; V˙  O2max, maximal oxygen uptake; VT, tidal volume; fb, breathing frequency; ΔHR, change in heart rate from rest to mimic; Te, expiratory time; Ttot, total breath time; PetCO2, end-tidal carbon dioxide tension; V˙  O2RM, respiratory muscle oxygen uptake; WOB, work of breathing; EfficiencyRM, efficiency of the respiratory muscles determined by dividing the measured V˙  O2RM by the calculated ideal oxygen uptake needed to perform the work;  ΔPoe, oesophageal pressure swing; WOBres, resistive work of breathing; WOBel, elastic work of breathing; EELV, end-expiratory lung volume; EILV, end-inspiratory lung volume; V˙  ECap, ventilatory capacity. * Significantly different compared to men (P<0.05).  Values are mean±SE. 50     How do our results for V˙  O2RM in men versus women compare to previous reports?  To date, sex differences in V˙  O2RM have been found by some (43, 131) but not all studies (81).  Eckermann and Millahn (1962) assessed ventilations up to ~120 l min-1 and concluded that women had a greater V˙  O2RM.  However, the absolute values for V˙  O2RM reported in their study were excessive (>2 l min-1) and do not, therefore, represent a realistic estimation.  Nonetheless, their estimation of respiratory muscle efficiency is similar to ours and others (2), and they noted that men may be more efficient.  Although more recent estimates of V˙  O2RM appear to be reasonable, there is still no consensus as to whether a sex-difference is apparent (81, 131).  A consideration when interpreting the two aforementioned studies is the narrow range of V˙  E investigated and the less than ideal accurate replication of exercise breathing patterns (i.e., lack of oesophageal pressure).  Specifically, both studies estimated the V˙  O2RM in women at a V˙  E ≤60 l min-1, which is approximately the threshold we found for a sex differences in V˙  O2RM.  Therefore, even with precise replication of exercise breathing patterns, a significant sex effect may have been masked by the low V˙  E.  A more suitable comparison for our data would be the work of Aaron et al (1992a,b).  As in the current study, those authors accurately mimicked exercise breathing patterns, matched the WOB and had subjects perform multiple trials across a wide range of V˙  E.  When comparing our male subjects to those of Aaron et al. (1992 a,b) (who tested 7 men and 1 woman), our absolute V˙  O2RM was similar and we both estimated that maximal V˙  O2RM accounts for ~10% of whole-body oxygen uptake.  The lone female subject in the previous study (3) had a V˙  O2RM that represented ~15% of whole body oxygen uptake, which is commensurate with our results for women (13.8 ± 1.5% of V˙  O2max).  Notably, one woman in our study had a V˙  O2RM representing 24% of  51  total oxygen uptake for two trials (Figure 10), which is remarkable considering others have shown that active muscle tissue can account for upwards of 85% of V˙  O2 (105).  While we note that 24% is on the upper limit of expected respiratory muscle oxygen uptake, others have reported similar values (3).  As expected, this subject developed EFL, used 85% of their ventilatory capacity and had an end-exercise V˙  E and WOB that was greater than the female average.  Thus, many of the predisposing factors associated with sympathetically mediated blood flow redistribution are present and there is evidence from animal models to suggest the diaphragm may be less sensitive to vasoconstrictor activity (1).  Given the above influencing factors and the repeatability of our measures, we are confident our values are physiological.  Overall, we conclude that women have a greater V˙  O2RM than men as a result of a greater WOB and decreased efficiency.  Mechanical ventilatory constraints.  To investigate the role of mechanical ventilatory constraints on V˙  O2RM, we categorized our subjects based upon the occurrence or absence of EFL during exercise.  Expiratory flow limitation occurs when maximal flow plateaus despite an increase in driving pressure (70) and can arise in young healthy subjects during intense aerobic exercise (12, 76).  The occurrence of EFL during exercise is associated with an increase in operational lung volumes (104), exacerbated exercise-induced arterial hypoxaemia (33) and reduced exercise performance (71).  Others have theorized that EFL is associated with a greater V˙  O2RM (3) but no specific data were presented to support this postulate.  Similarly, we found that those who developed EFL during maximum exercise had a greater V˙  O2RM (Figure 12).  In addition, our flow limited and non-flow limited groups had similar V˙  O2max, V˙  Emax and WOB.  At submaximal exercise, minimal if any EFL was present and, as such, there were no statistical differences between the groups for any variables (Figure 12).  Furthermore, there was an equal distribution of sexes and both groups successfully replicated their exercise breathing patterns.  Others have shown that aerobic fitness does not predict who does and  52  does not develop EFL (123) and we have argued that women develop EFL more often than men (53).  In the current study, however, we found an equal distribution of EFL between the sexes.  We intentionally recruited trained men in an attempt to ensure a similar distribution of flow-limited subjects in order to address our primary research question (regarding sex-differences in V˙  O2RM) in the most conservative fashion.  If we did not recruit trained men, we anticipated that few, if any, of the male subjects would have developed EFL and we would be less able to accurately discern sex differences in V˙  O2RM.  In all cases, subjects who developed flow limitation during exercise did so during the hyperpnoea trials, and vice versa for the non-flow limited subjects (Figure 12).  Therefore, variation in replicating spontaneous breathing patterns was not responsible for the greater V˙  O2RM noted in the flow limited group.      Figure 12. Composite average maximal expiratory flow-volume (MEFV) curves for subjects displaying expiratory flow limitation (EFL) (Panel A) and those with no expiratory flow limitation (NEFL) (Panel C). Placed within the MEFV curves are resting (thin solid lines) and the 100% V˙  Emax tidal flow-volume loops during maximal exercise and voluntary hyperpnoea.  There was no difference in V˙  O2max or V˙  Emax between the groups.  At 100% V˙  Emax the EFL group had a  53  greater V˙  O2RM, whether expressed as absolute (Panel B) or % of V˙  O2max (13.5 vs. 9.2 % V˙  O2max for the EFL and NEFL groups, P<0.05).  V˙  O2max, maximum oxygen uptake; V˙  Emax, maximum minute ventilation; V˙  O2RM, oxygen uptake of the respiratory muscles.  Values in Panel B are mean±SE. * significantly higher in EFL versus NEFL (P<0.05)  While we did not make anatomical estimations of airway size in the current study, those who develop EFL are thought to have smaller airways (35, 87).  Smaller airways are consistent with the greater resistive WOB noted at maximal exercise in the EFL subjects, despite the total WOB being similar between groups (Table 4).  As the maximal effective driving pressure for flow was approached, compression of the airways may have been initiated - a phenomenon termed “impending flow limitation” (87, 92).  The impending flow limitation could have altered breathing patterns via compression of airways, thereby resulting in a decreased efficiency of the respiratory muscles.  Other factors that could explain the greater V˙  O2RM in the EFL groups relates to those detailed above such as chest-wall deformation, abdominal stabilization and greater muscle shortening velocity from an increased breathing frequency.  The chest-wall deformation and abdominal stabilization could lead to a greater V˙  O2RM through additional muscular contraction, yet the work may not be accounted for, which is seen by our similar WOB values (Table 7).  54  Table 6. Cardiorespiratory variables during voluntary hyperpnoea at different percentages of maximal exercise ventilation for the EFL (n=10, 50% men) and NEFL group (n=8, 50% men).  45% V˙  Emax 60% V˙  Emax 75% V˙  Emax 100% V˙  Emax  EFL NEFL EFL NEFL EFL NEFL EFL NEFL % V˙  O2max 74±2 69±4 83±1 84±2 93±1 92±1 100 100 VT (l) 2.2±0.2 2.5±0.4 2.4±0.2 2.7±0.4 2.4±0.2 2.8±0.4 1.9±0.3 2.6±0.3 fb (breaths min-1) 32±2 28±3 37±2 35±3 44±2 43±3 60±2* 53±3 ΔHR (beats min-1) -3±3 2±3 8±2 4±3 10±2 6±2 24±3 19±3 Te/Ttot  0.56±0.01 0.54±0.01 0.56±0.01 0.53±0.01 0.54±0.01 0.53±0.01 0.53±0.01 0.52±0.01 PetCO2 (mmHg) 38±1 39±1 39±1 38±1 36±1 32±2 33±2 34±2 V˙  E (l min-1) 69±6 67±8 87±6 91±9 108±9 114±10 136±11 138±12 V˙  O2RM/ V˙  E (mlO2 l-1) 1.4±0.1 1.5±0.1 1.8±0.1 1.8±0.1 2.2±0.3 2.0±0.1 3.3±0.4* 2.4±0.1 V˙  O2RM/WOB (ml O2 J-1) 0.9±0.1 1.0±0.2 1.0±0.1 1.0±0.1 0.9±0.1 0.8±0.1 1.0±0.1 0.8±0.1 EfficiencyRM (%) 4.0±0.8 4.7±0.5 4.7±0.8 4.4±0.9 4.5±1.0 6.2±0.7 4.6±0.8* 6.2±0.5 ΔPoe (cmH2O) 23±1 20±2 28±2 28±3 35±2 37±3 52±3 49±3 WOB (J min-1) 114±15 108±25 168±19 183±33 265±33 296±45 485±63 443±67 WOBres (% total) 50±5 43±2 56±4 54±4 67±3 64±4 79±1* 72±2 WOBel (% total) 50±6 57±5 44±4 46±4 33±3 36±4 21±1 27±2 EELV (% FVC) 40±3 39±3 40±3 38±3 39±3 37±2 43±2 42±2 EILV (% FVC) 83±2 83±3 86±2 85±3 86±2 86±2 86±1 89±2 V˙  E/ V˙  ECap (%) 39±3 31±3 48±3 43±4 63±4 54±5 77±2* 63±3  EFL, expiratory flow limited; NEFL, non-expiratory flow limited; V˙  O2max, maximal oxygen uptake; VT, tidal volume; fb, breathing frequency; ΔHR, change in heart rate from rest to mimic; Te, expiratory time; Ttot, total breath time; PetCO2, end-tidal carbon dioxide tension; V˙  E, minute ventilation; V˙  O2RM, respiratory muscle oxygen uptake; WOB, work of breathing; EfficiencyRM, efficiency of the respiratory muscles determined by dividing the measured V˙  O2RM by the calculated ideal oxygen uptake needed to perform the work;  ΔPoe, oesophageal pressure swing; WOBres, resistive work of breathing; WOBel, elastic work of breathing; EELV, end-expiratory lung volume; EILV, end-inspiratory lung volume; V˙  ECap, ventilatory capacity. * Significantly different compared to NEFL (P<0.05).  Values are mean±SE.  55    Technical considerations.  We considered the possibility that respiratory muscle fatigue during the hyperpnoea trials could have affected our results.  High intensity exercise to exhaustion has been shown to induce respiratory muscle fatigue (73, 128), which may persist for up to 24 hr (79).  Respiratory muscle fatigue is associated with a progressive increase in muscle sympathetic nerve activity (32, 124), alterations in resting blood flow distribution (116) and reduced exercise capacity (57, 129).   Voluntary hyperpnoea has also been shown to elicit respiratory muscle fatigue (107).  Thus, it is conceivable that our subjects developed respiratory muscle fatigue and the associated neurovascular effects.  In the absence of heavy exercise, however, respiratory muscle fatigue only develops when the WOB is significantly greater than that achieved during maximal exercise (16).  In our study, the mechanical WOB did not exceed the maximal exercise values in any of the hyperpnoea trials.  Furthermore, the trials were relatively short and the subjects were provided with substantial rest between trials.  Finally, we found no effect of trial order on our estimates of V˙  O2RM.  If respiratory muscle fatigue was present, we would expect a systematic temporal change in the ability to replicate the exercise breathing pattern and/or V˙  O2RM.  Consequently, it is unlikely that fatigue developed and if it did, there does not appear to be any measurable effect on the V˙  O2RM.   During the voluntary hyperpnoea trials there was a significant increase in heart rate beyond 60% V˙  Emax.  As such, myocardial work likely contributed in small part to the observed increase in V˙  O2.  However, the increases in heart rate were similar for men and women and we presume therefore that cardiac V˙  O2 was also similar between the sexes.  Sex-based comparisons.  A difficult and often encountered problem when designing and interpreting studies regarding sex-based differences is how to best compare men and women.  Men, on average, are taller than women and therefore will have greater absolute lung volumes and flows.  Due to a greater muscle mass, men will generally  56  achieve a higher absolute V˙  O2 and V˙  E.  The principle issue here is whether to compare the sexes using absolute or relative values, a concern shared in other fields (58, 67).  In the current study, we made several comparisons in order to individually address specific questions and collectively provide an overall interpretation.    Another confounding variable when assessing sex-based differences is whether subjects should be matched for one or more anthropometric or functional variables (119).  For example, men and women could be matched for height, lung size, lung function, aerobic fitness or body composition.  However, matching may be best justified when attempting to isolate a single mechanism rather than attempting to understand the integrative whole-body response.  For example, women appear to develop exercise-induced arterial hypoxaemia to a greater degree than men (33, 55), but when matched for height (and consequently lung size) and aerobic fitness the gas exchange disparity is minimized (98).  Another example to illustrate the effect of scaling stems from the current study.  The woman with the lowest V˙  O2RM/ V˙  E was the tallest, had the largest lung volumes and greatest flows of all the women.  Despite her high aerobic fitness (60 ml kg-1 min-1), she did not develop EFL and her WOB was similar to the men.  As such, when the sexes overlap in anatomical variables, the physiological sex differences appear to be minimized.  However, we emphasize that many of the variables used to match men and women are themselves extensively influenced by sex.  Matching men and women for lung size may allow for certain comparisons, but it would eliminate a consistent and population-wide sex-based difference, rendering the results less generalizable.  Perspectives.  What are the implications of a greater V˙  O2RM in women on the integrative physiological response to exercise?  During maximal exercise in men, the WOB influences active leg blood flow and the distribution of total cardiac output through a sympathetically mediated response (54, 56).  As shown in Figure 11B, the slope of the V˙  O2RM-WOB relationship is significantly greater in women compared with men.  The  57  greater slope of the V˙  O2RM-WOB relationship in women indicates that for a given change in WOB, women have a greater change in the total V˙  O2 dedicated to the respiratory muscles.  Therefore, it could be hypothesized that when compared to men, women may show greater changes in leg blood flow when the WOB is altered by the same amount.  Further support for the above hypothesis arises from findings on anesthetized male and female rabbits.  Female rabbits dedicate a greater amount of blood towards the diaphragm in response to increases in ventilation elicited by hyperthermia (83).  A caveat to the idea that women may dedicate greater blood flow to the respiratory muscles is the greater β-adrenergic receptor activity in pre-menopausal women resulting in a blunted response to sympathetically mediated vasoconstriction (59).  To date, the influence of WOB on blood flow distribution during exercise has not been studied in women.  To accurately determine the potential effect of sex on WOB related alterations in blood flow, respiratory muscle work will need to be experimentally reduced while leg blood flow is directly measured. Previous authors have argued for the existence of a maximal effective V˙  E, defined as the ventilation beyond which further increases in external work would require the increase in V˙  O2 to be dedicated solely to the respiratory musculature (103).  In men, the maximal effective ventilation is significantly above V˙  Emax (3), but given the greater            V ˙ O2RM in women, we question if an effective ventilation could be attained.  We found that the      V˙  O2RM per V˙  E in women (Table 6) would need to be ~2.5x greater to equal the change in whole body V˙  O2 per V˙  E; men would require a value ~4x greater.  To attain the maximal effective V˙  E, the women would have to increase their maximal ventilation by 18 l min-1, which would result in significant hypocapnia (end-tidal carbon dioxide <20 mmHg) and would not be sustainable.  Using the alveolar gas equation, we estimate that the women’s V˙  O2max would have to be 63% greater, or ~4.5 l min-1 (~80 ml kg-1 min-1), for the greater V˙  E to be sustainable.  Similarly, for the men to achieve their maximal  58  effective ventilation, V˙  Emax would have to increase 40 l min-1 and V˙  O2max would have to increase by 67% or to ~98 ml kg-1 min-1.  Accordingly, we conclude that women may be relatively closer to their maximal effective V˙  E, but the corresponding oxygen uptake is only achievable in a small percentage of highly-trained athletes.    Conclusion.  Three primary conclusions can be drawn from our study.  First, at submaximal and maximal exercise intensities, V˙  O2RM is significantly greater in women compared to men.  Second, during heavy exercise, the V˙  O2RM represents a greater fraction of whole body V˙  O2 in women.  Finally, subjects who develop expiratory flow limitation during exercise have a greater maximal V˙  O2RM than those who do not develop flow limitation.  Overall, our findings indicate that the oxygen cost of exercise hyperpnoea is greater in healthy women than in healthy men, but neither sex readily achieves maximal effective ventilation.  The greater V˙  O2RM in women may have implications for the integrated physiological response to exercise.   59  Chapter Four: A proportional assist ventilator to experimentally unload respiratory muscles during exercise in humans.  4.1 Introduction In order to maintain appropriate alveolar ventilation in spontaneously breathing humans, some degree of work must be performed by the respiratory musculature.  The work of breathing (WOB) during eupnoea is minimal and represents a small fraction of whole-body energy expenditure (101).  However, during dynamic exercise the WOB increases exponentially as minute ventilation (V˙  E) rises and the respiratory muscle oxygen uptake can represent upwards of 15% of maximal O2 uptake (V˙  O2max) (3, 38).  To better understand the complex inter-relationships between ventilation, WOB and the bioenergetics of the respiratory musculature during exercise a long-standing approach is to experimentally manipulate the WOB.  An often-used method is to increase the WOB via an increased resistive or elastic load (27).  A less-utilized approach is to decrease the normally occurring WOB.  Decreasing the WOB during exercise is advantageous because it can provide insights into regulatory mechanisms that adjust to changing respiratory muscle work.   A common method to experimentally decrease the WOB during exercise is achieved by replacing nitrogen (N2) with helium (He) as the inspirate backing gas.  As these gases have different densities (He: 0.18 vs. N2: 1.25 kg m3 at STPD), a He-O2 inspirate increases the propensity for laminar flow, increases maximal flows and decreases the resistive WOB.  Helium has the added benefit of being technically simple, safe and easy to implement in a variety of experimental exercise settings in healthy and clinical populations (14, 33, 44).  However, there are some caveats when considering the utilization of He, as it reduces the WOB by variable amounts that depend on flow and is    60  relatively expensive to use for prolonged studies.  In addition, the effects of He are dependent upon the level of ventilation.  Helium minimally reduces the WOB when V˙  E is low, whereas, as ventilation increases the effect of He is amplified.  Finally, the exact mechanism behind the alleviation of mechanical constraints (such as flow limitation or high WOB) is not fully understood and could result from decreased WOB or increased flow via lessening of the Bernoulli effect (18).  Another method to reduce the WOB involves using a mechanical ventilator to provide a portion of the respiratory work.  With most mechanical ventilators (pressure or volume support) the degree of assist can be precisely controlled by the investigator.  However, most mechanical ventilators do not allow spontaneous breathing patterns and only provide a fixed amount of support which is significantly less than the flows and volumes achieved with heavy exercise.   An alternative form of mechanical ventilation is a proportional assist ventilator (PAV) (141).  A PAV amplifies the subject’s own respiratory effort, thus providing a dynamic level of inspiratory assist (expiratory assistance is possible, but less common).  The level of assistance can be high, where the ventilator provides the majority of work, or low, where the ventilator provides minimal support.  The primary advantage of a PAV is that the level of support is proportional to the subject’s own respiratory efforts, rather than predetermined set-points for respiratory timing, pressure or volume.  There are commercially available ventilators that are similar to PAVs that have been used in an exercise setting, but their utility is limited to relatively low exercise intensities.  For example, bi-level positive airway pressure support has been used to unload the respiratory muscles during exercise in heart failure (100).  However, these devices used can only support a V˙  E up to ~100 l min-1.  While this level of support is adequate for studies of clinical populations, it is insufficient for studies of healthy subjects performing heavy exercise.  The initial PAV (142) was able to successfully off-load the respiratory    61  muscles during mild-to-maximal (V˙  E>140 l min-1) exercise in young subjects (46).  The initial version of a PAV used a rolling seal spirometer driven by a linear actuator and was capable of unloading the respiratory muscles and resulted in a >50% reduction in WOB during intense exercise.  Unfortunately, sourcing or replacing many of the original parts for this ventilator is no longer possible and the original prototypes are no longer available. Experimental usage of a PAV has been instrumental to our understanding of the integrative aspects of the physiology of exercise (6, 54, 56, 110).  However, to the authors’ knowledge there are currently no usable PAV devices available to researchers in the field.  The goal of our manuscript is to describe how our version of a PAV remedies three shortcomings in the literature.  First, we describe the creation of a device to non-invasively unload the respiratory muscles across a range of ventilations in healthy subjects.  Second, we demonstrate how to construct a PAV device using contemporary components and computer software.  Third, we expand on the physiological response to assisted breathing in order better characterize the usage during exercise such that others in the field may use this experimental tool.   62  4.2 Material and methods Theory  The apparatus described in this manuscript uses the equation of motion in order to determine the level of assist.  The equation of motion and its usage with respect to the respiratory system has been described in detail elsewhere (18, 141), as such, we provide a brief description with specific application to our PAV.  The pressure required of the respiratory muscles (Pmus) to breathe is the sum of the elastic, resistive and inertial components.  The elastic aspect is the product of volume (V) and elastance (E), the resistive component is the product of flow (V˙   ) and resistance (R) and the inertial component if the product gas acceleration ( ) and inertance (I).  For the purpose of this manuscript, the E, R and I represent the values for the whole respiratory system.  The specific relationship is denoted in equation (1): Equation (1):      Equation 1 describes the pressure required during spontaneous unassisted breathing.  When some of the work is accomplished with a ventilator, the term for airway pressure (Paw) is added to the Pmus and provides the following equation: Equation (2):     It now follows that the pressure to produce a given volume and flow is the combined effort of the subject’s own musculature (Pmus) and the ventilator (which creates Paw).  Therefore, if the ventilator provides a greater amount of the work (i.e., an increased Paw) the respiratory muscles provide less work.  In practice, the PAV should provide only a portion of the total WOB and each parameter in Equation 2 must be accurately measured (or conservatively estimated) or there is a potential for a “run-away” system to exist.  When this occurs, the driving input pressure is greater than the subject’s opposing pressure to terminate the inspiration and the ventilator can continue to increase Paw    63  despite the subjects desire to expire (144).  At atmospheric pressure (or minimally above from the PAV positive pressure) and physiological breathing frequencies (<70 bpm), the component I is minimal (91) and is thus not included as an input parameter.  The distinguishing aspect of PAV is proportionality.  During an inspiration with a PAV, the subject’s own effort is amplified in proportion to the proportionality setting.  For example, if the PAV was set at 75% assist, the subject would have to provide 25% of the total work, and this is accomplished by a 3:1 proportionality setting where for every 1 unit of respiratory work performed by the subject the ventilator would provide 3.   Apparatus Description  As mentioned above, a PAV capable of performing during exercise has previously been constructed (142).  While the fundamental physical principles (equation of motion, proportionality) are similar, our apparatus differs significantly in that it utilizes compressed air to deliver pressure.  Using compressed air achieves the same goal (positive mouth pressure), as a rolling seal spirometer but all the hardware is commercially available.  Others have utilized a compressed gas system for usage during simulated resting conditions (82), but our PAV system has been designed and tested on subjects in order to accommodate high ventilations.  A schematic of the system is shown in Figure 13 where compressed gas tanks are regulated to between 140-480 kPa and connected to a proportional valve (Proval) (Standard PFCV 300 Series, IQ Valves, Melbourne FL).  The valve responds linearly to a voltage from the control software.  The output of the proportional valve is directed into the opening of a 25 L air-tight ballast tank.  The ballast tank serves to: (i) dampen the pressure wave, (ii) reduce acoustic noise from the Proval and (iii) can be partially filled with water to provide humidification and reduce deadspace.  Pressure within the ballast tank is measured through a side port connected to a manometer and excess pressure is vented through an adjustable bleeder    64  valve.  The exit port of the ballast tank is connected via large bore (40 mm i.d.) rigid pipe to a pair of rapid in-line on/off pneumatic controlled valves (Inspval) (KF25, Kurt Lesker, Vaughan, ON) that control airflow on the inspiratory side of the breathing circuit.  The valves are normally closed (via a spring) and open in response to compressed air delivered from a pair of 4-way solenoid valves (Solval) (T series, Versa Valves, Kalamazoo, MI) that act on the internal piston of the Inspval.  The pneumatic system operating the valves is fully sealed so none of the driving gas that acts on the Solval leaks into the breathing circuit.  As such, inspired and mixed expired gases remain pure and can be used in metabolic calculations.  From the Inspval, airflow is directed through a rigid pipe (40 mm i.d.) that contains several adjustable bleeder valves (PEEP Valves, Ambu, Columbia, MD) that prevent excessive pressure directed to the subject and potential barotrauma.  The distal end of the rigid tubing is connected to a “t” housing (model 2700 Hans Rudolph, Kansas City, Mo).  The “t” allows the compressed air to be delivered to the subject while also permitting “fresh” air to be inspired via one-way diaphragm valve.  The fresh air port is needed for the initial triggering of an inspiration and to provide subjects with additional air if they require more than is being delivered from the PAV.  However, when the PAV is adequately meeting the subject’s airflow needs, pressure within the housing is positive and the diaphragm valve is closed.  The third side of the “t” housing is directed towards the subject and passes through a pneumotachograph (model 3813, Hans Rudolph).  The airflow then moves through flexible large bore (50 mm i.d.) tubing connected to the mouthpiece (7200 Series, Hans Rudolph).  Mouth pressure (Pm) and end-tidal carbon dioxide are measured via ports in the mouthpiece.  The expiratory side is identical to the inspiratory side, with the same flexible large bore tubing connected to a pneumotachograph, which connects to rigid large bore pipe and two rapid in-line on/off valves (Expval).  The Expval are identical to the Inspval and are driven by the same Solval, but in an opposite configuration.  Through    65  rigid pipe (40 mm I.D.) the Expvalves empty into a mixing chamber, where mixed expired gases are sampled for metabolic calculations and then vented to the atmosphere.  Figure 13. Schematic representation of the proportional assist ventilator.  Drawing not to scale.  Abbreviations: E, subject elastance; Expval, expired valve; Inspval, inspired valve; K, proportionality constant; Paw, airway pressure; Pbal, ballast tank pressure; PetCO2, end-tidal carbon dioxide tension; Proval, proportional valve; PTI, inspired pneumotachograph; PTE, expired pneumotachograph; R, subject resistance; Safety, software safety parameters as described in the methods. Programing  All software was developed using LabView (LabView 2012, National Instruments, Austin, Tx).  Inspired and expired flows are digitally combined to create a dual flow channel.  By convention, inspired flow is designated as negative and expired flow as positive.  The software was designed to continuously monitor flow in order to detect negative or positive zero crossings.  A negative zero crossing represents the transition from expiration to inspiration and is characterized by flow becoming negative; with the opposite for a positive zero crossing.  The threshold and sensitivity of either zero    66  crossing can be adjusted independently.  Once a negative zero crossing is detected, the Solval configures to allow compressed air to flow into the piston of the Inspvalve, which opens it.  Simultaneously, the change in configuration exposes the piston of the Expval to atmospheric pressure, which reduces the internal pressure and causes the spring to shut the valve.  The opposite occurs during a positive zero crossing, whereby the Inspval are shut by their internal spring and the Expval open.  The valves are necessary on both sides of the breathing circuit in order for the development of positive pressure during inspiration.  Mouth pressure and ballast tank pressure are also continuously monitored and displayed.  Other ventilatory variables such as breathing frequency, tidal volume and ventilation are calculated and available in real-time.  When unloading is desired, the system is set to the PAV function.  During the PAV mode, the system works to match instantaneous measured Pm with a theoretical unloaded positive Pm.  The theoretical unloaded Pm uses the proportionality constants, real-time flow and volume along with adjustable values for E and R all inputted into the equation of motion.          Safety features  Several features were implemented to ensure subject safety during unloaded breathing.  Broadly, the features can be divided into physical and programming features.  Physical features operate in the absence of any programing or electric input.  Specifically, several bleeder valves are located in the breathing circuit to ensure the subject is not exposed to excessive pressure.  Furthermore, the normal configuration of the Proval and Inspval is closed, and the Expval is open; thus in a failure situation, the default configuration prevents the subjected from being exposed to high pressure and unimpeded breathing is permitted.  Programming safety features are as follows.  (i) An adjustable maximal allowable tidal volume.  The adjustability is to ensure an appropriate maximal    67  limit for subjects of varying size.  (ii) An upper limit for maximal Pm.  If either situation (i, ii) is achieved, the system immediately ceases unloading and configures to the exhalation position. (iii) A minimal expiratory time must be achieved before an inspiration can occur.  This prevents the system from starting to unload in a “non-breath” situation.  For example, if a subject coughs, a rapid change in flow occurs and this may be detected as a zero crossing and result in the system in a wrong configuration.  If the minimal expired time is not met, the system stays in the passive expired conditions, during which inspiration can occur unimpeded through the free air valve and unopposed expiration through the Expval.  The pitfall of this feature is that a breath can go undetected, whereby a true inspiration occurs, but the system does not unload; however, normal function is resumed after the missed breath. (iv) The order of sequence must be inspiration followed by expiration.  The program will not allow a second unloaded inspiration to occur without an adequate expiration in between.  The result when this condition is met is identical to the above, whereby unimpeded breathing is allowed for a single breath. Breathing Simulator  Before performing human trials during exercise, a breathing simulator was created in order to simulate some features of exercise.  Briefly, an electric motor engaged several gears, the last of which was connected to a spinning lever arm.  The lever arm was connected to the plunger on a 3 L calibration syringe.  As the lever arm rotated, it depressed and withdrew the syringe plunger.  The distal end of the calibration syringe was attached to the mouth piece of the PAV.  The tidal volume of the simulator could be adjusted by moving the plunger attachment on the lever arm.  One full rotation took ~ 1000 msec, which corresponds to a breathing frequency of 60 bpm; a common maximal rate during heavy or maximal exercise.  The electric motor draws a constant voltage (115    68  V) from its power source and varies the amperes based on work.  As voltage is constant, the amperes drawn are directly proportional to the work performed and are analogous to Pmus in Equation 1.  During operation the amperes drawn to the motor were continuously monitored (AT5 B10, LEM, Milwaukee, WI).  When unloading, the PAV should accomplish some part of the mechanical work.  If the external work of the apparatus is maintained (similar tidal volume and frequency), the motor has less work to accomplish and this would be detected by a decrease in amperes needed.    69   Figure 14. Raw traces of flow (Panel A), mouth pressure (Panel B) and power consumption by the motor driving the piston (Panel C) from the breathing simulator. For both the PAV (blue) and control (red) condition the breathing frequency was 60 bpm and the tidal volume was ~1.25 L.  Each condition has 5 breaths superimposed and aligned.  The amperes in Panel C has been referenced to the minimal power to overcome frictional and inertial resistance (~0.47).  As such, the negative region in Panel C does not represent power gained by the motor, but rather the PAV is accomplishing all the work to    70  move the piston and some of the frictional and inertial resistive work.  In Panel C, the light shaded area represents the power needed when the PAV was engaged.  The sum of the dark and light shading is the power needed during the control condition.  The stippled area is not included in the calculation of the power consumption For the trials described below, the breathing simulator was set to a frequency of 60 bpm with a ~1.25 L (ambient temperature, pressure) tidal volume, resulting in a ~75 l min ventilation.  When running the breathing simulator for 10 minutes, the PAV failed to detect 0 breaths, indicating that in the absence of noise introduced from human subjects (e.g., coughing, clearing throat, etc.) our breathe detection system is effective.  Figure 14 shows flow, Pm and amperes drawn from the electric motor for 5 breaths on the PAV and 5 serving as a control with the apparatus on and no unloading occurring.  The pattern of inspiration is similar between conditions with comparable values for overall tidal volume and breathing frequency and as such the external work is nearly identical.  In Panel B, at the initiation of a breath the Pm for both conditions is identical as the PAV requires an initial negative flow (consequently negative pressure) to detect a breath.  Thereafter, the PAV engages and Pm becomes positive in proportion to flow and volume.  As the breathing simulator had no elastance and minimal resistance, the PAV parameter were set to values that would achieve similar unloading seen by others (142).  With the PAV accomplishing part of the external work (by creating positive Paw), the amperes drawn, and thus the motor’s work, is reduced throughout the entire inspiration (Panel C).  However, after the inspiratory phase is over, all parameters are identical between conditions, indicating that the PAV is not hindering expiration.  On average, the amperes drawn by the motor were reduced to 23% of the control value (average 0.0311 ± 0.0012 vs. 0.0072 ± 0.0003 A∙s-1, for control and PAV respectively).  The stippled area was not included in the calculation as the amperes drawn were below the initial current needed to initiate motion.  As such, the electric motor was not storing power, but rather the PAV was performing all the work to move the plunger and some of the work to overcome the    71  initial frictional and inertial resistance to movement.  The above phenomenon is akin to when oesophageal pressure (Poeso) is elevated above end-expiratory pressure while inspiration is still occurring during unloading.  In the human example, the PAV is performing all of the work to produce airflow and some additional elastic work; a visual example in humans is in Figure 15 and has been shown by others using a PAV (54, 110).   Satisfied that the apparatus could consistently detect zero crossings at a physiologically relevant tidal volume and frequency, properly regulate the on/off valves and reduce the work performed by the electric motor, we proceeded to trials with spontaneously breathing exercising humans.   Human Trials  Trials of the PAV involving human subjects were approved by the Clinical Research Ethics board at the University of British Columbia, which adheres to the Declaration of Helsinki.  All subjects provided written informed consent before participation.  No adverse effects from breathing on the PAV were reported by any subject.  All subjects were free from cardiovascular, pulmonary, and neurological disease, were non-smokers and regularly engaged in physical activity.  Subjects included endurance trained and recreational athletes of both sexes.  Experimental PAV trials ranged from intense (~90% V˙  O2max) constant-load exercise for >10 minutes to varying intensity (50-80% V˙  O2max) and shorter duration (2-5 minutes) discontinuous exercise. Work of breathing quantification  In order to quantify the effect of a PAV, an accurate assessment of the WOB is needed.  The two common methods to analyse and quantify the WOB during exercise are (i) integrating a composite average pressure-volume loop and (ii) the pressure-time product (PTP).  Making “Campbell” diagrams (22) is also common, but requires knowledge of lung volumes, which is not possible in our iteration of the PAV.  To    72  accurately determine the WOB, some adjustments to the techniques are required when using a PAV.  In all the described instances, the pressure utilized is Poes, rather than transpulmonary (Pm-Poes), which would erroneously overestimate the true pressure from the high Pm.  When using the pressure-volume curve, typically the area within the loop (and an additional elastic component that falls outside) is integrated and the sum multiplied by breathing frequency.  With addition of a compliance line connecting end-expiration and end-inspiration the total work can be partitioned (40).  A concern with this technique when combined with the PAV is that the end-inspiratory pressure can exceed end-expiratory pressure, resulting in a paradoxical compliance line.  Thus, the ability to partition the WOB into elastic and resistive components is not possible.  Accordingly, when determining the WOB with the PAV we integrated the area within the pressure-volume loop and did not attempt to separate the constituent points. To determine the PTP, pressure is typically integrated with respect to time.  To determine inspiratory work, oesophageal or trans-diaphragmatic pressure can be integrated throughout inspiration.  For expiratory work gastric pressure is integrated during expiration.  As mentioned previously, with high amounts of unloading, end-inspiratory pressure can exceed or become less negative than end-expiratory pressure.  This situation appears illogical because inspiration is still occurring (volume is getting larger) yet, pressure is similar or greater than at the beginning of inspiration.  When Poes during inspiration exceeds the initial inspiratory pressure, all the work is being accomplished by the ventilator.  In this situation Poes should not be integrated and used for the total estimation of WOB.    73  4.3 Results and discussion Representative data  Figure 15 depicts pressure and flow signals from a single subject exercising on a cycle ergometer at ~70% of V˙  O2max while breathing on the PAV during assisted and unassisted breathing.  The figure illustrates the transition from assisted breathing (left side) to unassisted breathing (right side) and shows the temporal response in key variables.  The principal mechanism for unloading can be seen in Panel A.  During no assist, Pm changes as expected during a normal respiratory cycle, with a negative deflection during inspiration and a positive during expiration. The absolute magnitude of the deflection is related to the resistance of the apparatus.  However, during assisted breathing, Pm is negative for a very brief moment (~20-40 msec) at the onset of inspiration (which is necessary to trigger the device) and is positive thereafter.  The faint hashed line in Panel A is the theoretical Pm (as determined by the equation of motion) necessary to produce the set level of assist; in this example ~75%.  Notice how the actual Pm minimally lags behind the theoretical Pm but shows a similar shape, an observation shown by others using a similar PAV but with a different pressure generating mechanism (spirometer) (46).  The lag is because of the software and mechanical delay and is beneficial as it decreases oscillations around the desired set-point pressure.  If the level of assist was increased (>75% assist) then the theoretical target would have a greater absolute pressure.  At the onset of expiration, Pm falls rapidly, but remains positive due to the unimpeded expiratory phase.  In Panel B, Poes is greatly reduced during assisted breathing, thus reducing the overall WOB despite similar V˙  E.  The transdiaphragmatic pressure (Panel C) is also reduced during assisted breathing, indicating less diaphragmatic work.  Although the PAV settings are static for this data, there is still    74  variability in the unloading on a breath-by-breath basis.  Minor variations in upper respiratory tract muscle tone and/or a willingness to “accept the unloading” results in Poes   Figure 15. Raw respiratory pressures and flow from a subject breathing on the apparatus with the PAV assisting (left of the solid vertical line) and not providing assist (right of the solid vertical line).    75  The subject was exercising on a stationary bike with a V˙  E of 89 and 90 l min-1 The solid vertical line is when the assisting function was turned off and the data represents a continuous series.  The shaded area indicates the time for two separate inspiration.  The solid line in Panel A is the actual mouth pressure, while the hashed line represents the theoretical unloading target as determined by the equation of motion.  variability between breaths despite similar flows (143).  Furthermore, our version of PAV has many built-in safeguards.  These safeguards can dampen and do not allow rapid changes in Pm, which result in varied Poes.  If the safeguards were not in place, Pm would rapidly oscillate around the target and could result in excessive pressure or volume delivery.     76   Figure 16. Oesophageal pressure throughout an inspiration during assisted breathing (PAV) and the control condition for a single subject exercising at different intensities on a cycle ergometer. Panels A, B, and D, show inspirations at fixed external workloads, whereas, Panel C depicts two external workloads that resulted in a similar V˙  E.  fb, breathing frequency; V˙  E, minute ventilation; PAV proportional assist ventilation.    77   Figure 16 shows the effect of increasing the external work, therefore greater V˙  O2 and V˙  E, and the ability of the PAV to assist breathing in a single subject.  At the onset on inspiration, Poes during both conditions is nearly identical and follows a similar path in all examples.  However, after ~0.2 sec, the Poes of the PAV supported subject is reduced at all time points, despite the greater V˙  E in this iso-work example.  The 0.2 sec delay is the result of software and valve speed delays (~20-50 mses) and the time it takes for the pressure wave to be generated and delivered.  This delay can be shortened by reducing deadspace in the apparatus and/or altering the sensitivity/threshold of zero crossing detection.  With higher V˙  E, and especially greater breathing frequencies, these additional steps are necessary to ensure adequate unloading and can be accomplished prior to beginning exercise or manually during exercise.   In all the PAV examples, the end-inspiratory Poes is greater than the initial pressure.  As such, when the PTP was quantified in these examples, the baseline was set to the initial pressure and anything above was not integrated.  For the iso-work examples (Panels A, B, D) the work performed by the subjects using PAV was 39, 56 and 68% of control for the 220, 240 and 270 W trials.  Although the level of unloading appears to be smaller as work increases, in each example the V˙  E is greater in the PAV conditions.  In Panel C, two different external work trials were used with similar V˙  E, demonstrating the greater degree of unloading iso-ventilations.  In this case, the work for the PAV condition was 36% of the control. Reduction in work of breathing  The ability of the PAV to reduce the WOB is shown in Figure 17.  Shown in Panel A are WOB values from integrating composite average oesophageal pressure-volume loops in a single endurance-trained male subject (i.e., high workload and sustained ventilation (>175 l min-1)).  By design, the WOB during assisted breathing on the PAV is lower than the control conditions.  Factors unrelated to the PAV could be    78   Figure 17. Panel A, work of breathing values from integrating oesophageal pressure-volume loops at different ventilation.  All data is from a single male subject completing different exercise tests within 2 months.  Panel B, work of breathing from pressure-time products for three constant load time-to-exhaustions exercise tests performed for identical time and work in a single female subject. During the “high” unload condition, the PAV was set to the highest tolerable degree of unloading.  Conversely, during the “low” unloading, the PAV was set to only minimally (<25%) unload the respiratory muscles.  Note, during PAV days, the work of breathing is still reduced despite an increased ventilation.  PTPoes, oesophageal pressure-time product; TTE, time-to-exhaustion; VE, ventilation. responsible for the lower WOB, however this is unlikely for several reasons. First, at a given V˙  E, the WOB is consistent between experimental days and exercise paradigms (Figure 17).  Specifically, the filled-in symbols all overlap and are from a single subject performing a maximal cycle test (circles) or two time-to-exhaustions test (diamonds, squares).  As such, if spontaneous breathing is allowed, there is minor within-subject variability from day-to-day testing or between protocols.  Therefore, the reduction in WOB seen with the PAV is not an effect of normal variation or different exercise testing protocols.  Second, the reduction in the WOB from the PAV is not a function of different external apparatus resistance.  The half-filled diamonds are WOB values when the    79  subject was breathing on the PAV, but with the level of assistance set to zero.  As the WOB values are similar between these points, the reduction in the WOB is not due reduced resistance from the PAV tubing.  Figure 17B shows oesophageal pressure time product derived work values from a single female subject performing three exhaustive exercise tests.  The tests were of similar duration and intensities but with three conditions: control, PAV with high unloading and PAV with low unloading.  During the high unloading condition, the WOB was reduced to ~30-55% of control values throughout the duration of exercise despite average minute ventilation that was 17 l min-1 greater.  During the low unloading condition, the WOB was still reduced, but to a lesser degree.  While it is clear that the PAV can vary the degree of unload, a specific desired response is difficult to achieve.  During the high unloading condition, the target was 60% unloading (resulting in WOB equal to 40% of control), but the actual unload was variable.  Given this, studies requiring reproducibly specific fractions of unloading may be difficult.   Previous studies using a PAV during exercise have reported a reduction in WOB ranging from 40-80% (6, 54, 110, 136).  The specific amount of unloading (as a % reduction in WOB) is influenced by both the PAV’s assistance setting and also the % of maximal V˙  E.  For example, the subject in Figure 17A has a maximal VE ~200 l min-1.  At V˙  E below 140 l min-1 (~70% of maximum V˙  E) the degree of relative unloading is greater (~70-80% unload) than at V˙  E, > 140 l min-1 (~50% unload) (Figure 17A).  This phenomenon can be explained by the constituent components of the WOB.  At a lower relative V˙  E, the majority of the WOB is needed to overcome inspiratory work.  However, as a subject approaches maximal V˙  E, breathing is relatively more rapid, expiratory muscles become increasingly active and consequently expiratory work contributes a greater fraction of the total WOB.  Currently, our PAV only unloads inspiration and    80  leaves expiration unimpeded.  Therefore at near maximal V˙  E, we are still able to unload inspiratory work to a similar degree as lower with V˙  E, but the expiratory aspect becomes a relatively greater part of the total WOB.  The result is an inflection point where the WOB during the PAV rises in a similar manner to that of unassisted breathing.  The inflection point in this example (Figure 17A) is ~140 l min-1 and also corresponds to when the increase in V˙  E is principally frequency driven, which only further increases the expiratory work from more forceful expirations and consequently greater flows.  It is important to note that the inflection will occur for different subjects at different absolute V˙  E.  For example, healthy women will generally have a lower maximal V˙  E than an age and fitness matched man (37, 118).  Therefore, the absolute V˙  E at which the PAV is not able to unload the WOB as much would occur considerably lower (~80 l min-1) in women.  Nonetheless, we found no difference in the % reduction of WOB between the sexes.  If the expiratory work also requires substantial reduction, the solution could be to use heliox (79% He:balance O2) gas rather than compressed room air for the PAV; a strategy employed in other PAV iterations (7) and tested in our apparatus with a 85% reduction in WOB.    Nonetheless, we stress that the above examples are from single representative subjects and the variability in WOB reduction can be large in a group.  For example, in a group of healthy subjects (n=8), the reduction in WOB was 42±12 (range: 10-60%) for a similar near-maximal exercise test.  Moreover, the variability may be greater when investigating other cohorts; for example, healthy aging or illness.   Whole-body effects   The goal of the PAV described in this study is to decrease the WOB during exercise.  On the other hand, the use of a PAV is often desired in order to investigate the integrated physiological response to exercise.  For example, if the WOB is reduced, and presumably the oxygen demand of the respiratory muscles is decreased, does this equate    81  to measurable differences in whole-body V˙  O2?  To verify this we tested a subject whose oxygen cost of breathing  we had previously determined (38) and had them exercise for a fixed duration and time with and without the PAV.  The average V˙  E for the trial without the PAV was 140 l min-1, which, based on the subject’s measured WOB corresponded to a V˙  O2RM of 262 ml min-1.   When the subject completed the identical exercise trial on the PAV, V˙  E was similar (145 l min-1) but WOB was reduced. With unloading,V˙  O2RM was estimated to be 159 ml min-1, 103 ml min-1 less than without PAV .  Accordingly, average whole body V˙  O2 for the control and PAV trial was 3.39 and 3.3 l min-1 respectively, or a 90 ml min-1difference, which is similar to the estimated difference in V˙  O2RM.  The consistent reduction in V˙  O2RM was preserved when we used values for the same subject during the final 30 sec of the trial when their V˙  E was greater (~190 l min-1).  In this case, based on the reduction in WOB, the V˙  O2RM would be ~300 ml min-1 and indeed whole body V˙  O2was 3.68 l min-1 rather than 3.90 l min-1 in the control condition.  A similar finding was shown by others who used V˙  O2RM values based on previous literature (136).     Methodological and physiological considerations  The PAV we have described requires a clean flow signal in order to accurately detect zero crossings and to trigger the ventilator.  When using a cycle ergometer the flow signal contains fewer movement artefacts as the subject is stationary compared with running.  During treadmill running the movement of the person, tubing and the foot-strike artefact all introduce noise into the flow signal.  Our description of a PAV was only tested during cycle ergometry.   The sensation of positive pressure at the mouth during inspiration on the PAV is considerably different than that of spontaneous negative pressure breathing.  In order to achieve optimal unloading, a subject must be willing to accept the ventilatory support provided.  In doing so, the subjects become more “aware” of their breathing.     82  Specifically, the subject must be coached to not dampen the inspiratory assistance and relax their respiratory muscles.  If not, subjects may paradoxically contract their respiratory muscles which will decrease the assistance from the PAV.  Accordingly, each subject must be given ample familiarization trials before an experimental trial.  Without exception, all subjects practised breathing on the PAV during exercise multiple times before we performed an experimental trial.  Unfortunately, due to the sensation of positive pressure and the practice required, it is not possible to blind the subject to the intervention.  Furthermore, the rigorous coaching and insistence that subjects relax and accept support could be viewed as a form of psychological preparation or priming.  Previous studies have shown that dyspnoea (the sensation of breathlessness) can be influenced by psychological training and current mood in both healthy and clinical subjects (80, 115, 132).  Accordingly, we suggest that interpreting differences in dyspnoea during exercise on the PAV should be done cautiously.  Any difference in dyspnoea may be influenced by confounding variables and the lack of blinding means the placebo effect cannot be excluded.  Finally, although it is speculative, this coaching and training has the potential to alter central command aspects of the respiratory system.  As such, the PAV may not only change respiratory mechanics, but alter the control of breathing. As mentioned above in the “work of breathing quantification” section, additional considerations are necessary when quantifying the WOB during PAV.  Specifically, the PAV creates instances that do not occur with normal spontaneous breathing.  Ultimately, the choice of methods for quantification depends on the question being addressed.  If the reduction of only inspiratory muscle work is needed, using PTP would be the best choice.  However, if the principal question requires the quantification of the reduction in total WOB, then integrating pressure-volume loops would give a better indication of the total    83  work done on the lungs.  Finally, the above methods to quantify the WOB are still a conservative estimate as work stabilizing the abdomen and distorting the chest-wall are not accounted for (47, 49).  Often these effects are underappreciated during normal exercise and the use of a PAV has the potential to increase this work even more. In our description of a PAV, it is difficult to determine lung volumes. For example, the inspiratory capacity method risks barotrauma when providing assist at high lung volumes.  The inert gas dilution technique is technically difficult because the subjects would have to breathe a known amount of precise gas while still delivering pressure support.  Even if the apparatus could be designed appropriately, the potential for error would result in low confidence of the results.  The final method with some potential would be to use optoelectronic plethsymography.  However, our PAV device blocks the necessary clear views of the thorax and abdomen during exercise.  Utilizing a PAV during exercise creates physiological effects which may or may not be desired, but must be considered when designing a study.  Chiefly, there are heart-lung interactions that occur due to the: alterations in intrathoracic pressure changes, effect of positive pressure ventilation in of itself, alteration of breathing patterns or respiratory muscle recruitment and changing metabolic demand.  Furthermore, the phase of respiration affects both sides of the heart differently (72).  Specifically, inspiration can augment right ventricular function via increased preload and simultaneous reduce left ventricular stroke volume (99).  A final caveat when interpreting heart-lung interactions is that most work is performed on quadrupeds or anaesthetized humans.  As such, extrapolating findings to a healthy human performing vigorous exercise may not be justified.  Accordingly, below we detail the experimental results involving heart-lung interactions during exercise in humans; and stress the complexity of the inter-relationship.      84  When unloading the respiratory muscles, the WOB is reduced and intrathoracic pressure swings are lessened (Figure 14, 15).  Independent of a PAV, altering intrathoracic pressure swings has been shown to impact venous return and result in a reduced cardiac output (96).  During near-maximal exercise with assisted breathing using a PAV, pulmonary vascular resistance (PVR) is increased despite a decrease in cardiac output, as compared to a iso-workload control (56).  The rise in PVR is most likely the result of relatively higher (less negative) intrathoracic pressure during the PAV which will compress capillaries or stretch other vessels.  However, changes in PVR could also be the result of breathing at higher lung volumes which independently can influence pressure (138).  Furthermore, although not statistically significant, when exercising on the PAV, stroke volume was reduced in all subjects compared to the control (56).  While it is clear that the PAV does affect the cardiovascular system, it must also be recognized that the whole-body V˙  O2 is lower with PAV than without, which may explain some of the decrease in cardiac output and stroke volume.  A final consideration is that PAV allows breath-by-breath variation in intrathoracic pressure.   Consequently, the mechanical interactions will be constantly changing.  While the exact mechanism and effect of PAV induced changes in cardiac function is complex and not well understood, it must be considered when interpreting results.   Future direction  The ability to reduce the WOB at all ventilations allows for different investigations into cardiorespiratory control.  For example, studying healthy subjects known to differ in pulmonary mechanics such as the sexes (117), normative aging (74) and clinical models such as chronic heart failure (100) or pulmonary rehabilitation (44).  It is known that pulmonary mechanics effect cardiac function (95) but the majority of this work is done in healthy subjects is at rest.  The PAV could be useful in manipulating    85  pulmonary pressure during various intensities of exercise and assessing the cardiac consequences.  Finally, altering the duration and degree of unloading could prove insightful as to when respiratory muscle fatigue occurs or the difference between cumulative or maximal rate of respiratory muscle work. Conclusions We have demonstrated that a PAV can be constructed of readily available parts and can be used to successfully unload the respiratory muscles during all V˙  E during exercise.  During operation, the PAV was able to create positive pressure during inspiration, which in turn decreased the respiratory muscles’ contribution to the total work.  When unloading the respiratory muscles, the increase in Paw resulted in quantifiable changes in the WOB and whole-body V˙  O2.  Accepting the unloading during exercise requires considerable practice and heart-lung interactions need to be considered.  In conclusion, the PAV is a valuable experimental tool to manipulate the WOB during exercise and study the ensuing effect. 86  Chapter Five: Reducing the work of breathing during intense exercise attenuates locomotor fatigue to a greater extent in women 5.1 Introduction In healthy young men, the respiratory system can influence the development of locomotor muscle fatigue during heavy exercise.  Specifically, normally occurring exercise-induced arterial hypoxaemia (EIAH) in healthy men has been shown to contribute to quadriceps muscle fatigue.  Relieving EIAH by breathing a hyperoxic inspirate attenuates the severity of muscle fatigue (109).  Furthermore, when the work of breathing (WOB) is minimized, locomotor muscle fatigue is also attenuated (110).  The reduction in locomotor muscle fatigue resulting from both manipulations (i.e. hyperoxia or reduced WOB) is thought to result from increased oxygen delivery to the working muscles.  When EIAH is reversed with a mildly hyperoxic (~26%) inspirate, oxyhaemoglobin saturation (SaO2) remains higher and consequently arterial oxygen content (CaO2) and delivery are improved.  When the WOB is reduced, blood flow is redirected towards the active locomotor muscles (54) via the respiratory metaboreflex, a sympathetically-mediated reflex originating in the respiratory musculature (30).  The effect of respiratory muscle work on blood flow redistribution is only thought to be elicited during intense exercise (>85% of maximal oxygen uptake (V˙  O2max)) when the WOB is high (136) and there is sufficient competition for the finite cardiac output (21).  Increasing oxygen delivery is thought to attenuate fatigue by enhancing Ca2+ release and uptake at the sarcoplasmic reticulum (42) and by lessening metabolic by-products such as H+ (4), lactate (61) and inorganic phosphate (63).   Compared to healthy men, height matched women have smaller lungs, airways and fewer alveoli (90, 130).  Even when the sexes are matched for absolute lung size, women still appear to have smaller conducting airways (120).  As a consequence of these anatomical differences, some aspects of the physiological response to acute whole-body exercise differ  87  between the sexes.  For example, for a given V˙  E, women have a greater total WOB (53, 134), caused by a greater resistive rather than viscoelastic work (37).  As a consequence of the greater WOB, women have a greater oxygen cost of breathing (V˙  O2RM) for similar absolute V˙  E (38).  Importantly, the respiratory muscles in women demand a greater portion of whole-body V˙  O2 at maximal exercise (38), and based on the Fick equation, it would suggest that women’s respiratory muscles would also command a greater fraction of total cardiac output.  Similar to men, women can also develop significant EIAH during intense exercise (33, 55).  However, unlike men, moderate and highly trained women can both develop EIAH partially due to mechanical ventilatory constraints (33, 87) and even non-aerobically trained women can develop EIAH (34). Given the known effects of EIAH and high WOB on locomotor fatigue coupled with the aforementioned sex-differences in the physiological response to exercise, we sought to address two questions.  First, to what extent does eliminating EIAH attenuate quadriceps muscle fatigue in both sexes?  Second, will reducing WOB during intense exercise attenuate quadriceps muscle fatigue similarly in both sexes? We hypothesized that, regardless of sex, the subjects who developed the lowest SaO2 during exercise would have the greatest attenuation in quadriceps fatigue when EIAH is reversed.  Furthermore, when WOB is attenuated, women will show a greater attenuation in quadriceps fatigue.  5.2 Methods Subjects.  After providing written informed consent, 16 healthy subjects (n=8 men) participated in the study.  All procedures adhered to the Declaration of Helsinki and were approved by the Clinical Research Ethics Board at the University of British Columbia.  Subjects had a range of exercise participation (recreational to national calibre athletics), did not report any current or previous cardiorespiratory ailments and had spirometry within  88  normal limits (11).  Previously, our laboratory has demonstrated significant inter- and intra-subject variability with respect to hormone concentrations throughout the menstrual cycle, but with no effect on submaximal exercise ventilation (84).  Therefore, we tested female subjects at random points throughout their menstrual cycle and oral contraceptive use was not an exclusion criterion. Experimental Design.  Subjects completed four testing days (Days 1-4) that were each separated by >48hrs.  Day 1 was a maximal exercise test to determine workload while Days 2-4 comprised of a time to exhaustion test (TTE) and fatigue measures.  On Days 2-4, after surface electromyogram (EMG) placement, baseline quadriceps muscle function was assessed.  Next, oesophageal (n=16) and gastric balloons (n=4) were placed and baseline diaphragm function was determined in a subset (n=4, n=2 women).  A TTE test was then completed, with fatigue assessed 3, 10, 30 and 60 min post.  Data collection was similar between all three TTE days; except for temperature-corrected arterial blood gas analysis being performed on Day 2.  The TTE on Day 2 served as a control, and determined exercise duration for subsequent days.  The TTE on Days 3, 4 involved the experimental manipulations and was performed for an identical time and intensity as Day 2, ensuring similar external work between days.  On one of these final two days, EIAH was reversed by breathing a hyperoxic inspirate, and on the other day the WOB was minimized by breathing with a proportional assist ventilator (PAV).  The order of manipulations on Days 3 and 4 was in random order. Maximal exercise (Day 1).  To obtain maximal work rate and baseline WOB values, a step-wise incremental test on a cycle ergometer (VeloTron Pro, RacerMate, Seattle, WA) was performed to the limit of tolerance after insertion and placement of an oesophageal balloon-tipped catheter.  Men began at 120 W and women at 80 W, with a 20 W increase  89  every 2 min for both groups.  Cardiorespiratory variables were assessed using customized hardware and software as described elsewhere (35). Table 7. Baseline variables and maximal exercise data  M W Age (years) 29±3 28±2 Weight (kg) 74±2 60±2* Height (cm) 179±2 166±3* FVC (l) 5.4±0.2 4.0±0.2* % pred 98±4 101±5 FEV1 (l) 4.4±0.2 3.2±0.1* % pred 98±3 96±3 FEV1/FVC (%) 82±3 81±3 Maximal exercise, Day 1   Workload (Watts) 338±17 235±7* HR (beats min-1) 186±3 185±4 V˙  O2 (ml kg-1 min-1) 60.5±1.6 52.7±2.7* V˙  O2 (l min-1) 4.5±0.2 3.1±0.1* V˙  O2 (% pred) 134±5 139±8 V˙  CO2 (l min-1) 4.7±0.1 3.3±0.1* RER 1.09±0.02 1.05±0.02 VT (l) 3.2±0.2 2.1±0.1* fb (beats min-1) 57±3 59±4 V˙  E (l min-1) 180±6 120±5* V˙  E/ V˙  O2 40±1 39±1 V˙  E/ V˙  CO2 38±1 37±1 EELV (% FVC) 34±2 38±2 EILV (% FVC) 86±1 87±1 ∆Poeso (cmH2O) 56±4 47±4 WOB (J min-1) 625±42 366±42* PTP (cmH2O l-1 sec-1) 595±35 552±50 V˙  ECap (l min-1) 212±7 160±9* V˙  E/ V˙  ECap (%) 85±1 76±5 EFL (n) 6 5 EFL (%) 26±1 21±5 Values are mean±SEM.  Abbreviations: M, men; W, women; FVC, forced vital capacity; % pred, percent predicted values; FEV1, forced expiratory volume in 1 second; HR, heart rate; V˙  O2, oxygen uptake; V˙  CO2, carbon dioxide output; RER, respiratory exchange ratio; VT, tidal volume; fb, breathing frequency; EELV, end-expiratory lung volume; EILV, end-inspiratory lung volume; ∆Poeso, oesophageal pressure swings; WOB, work of breathing; PTP, pressure-time product; V˙  ECap, ventilatory capacity; EFL, expiratory flow limitation defined as %  90  overlap of tidal breaths on the maximal expiratory flow volume curve. *, significantly different from men P<0.05  Time to exhaustion.  For all TTE tests we used the same cycle ergometer, maintained similar seat height/handle bar position and ensured cadence was similar.  Subjects refrained from adopting a “standing” position at any time during any of the tests.  On Day 2, subjects performed a self-selected warm-up and on Days 3, 4 the warm-up was for similar duration and intensity.  The exercise intensity was >85% of the maximal work achieved during the V˙  O2max test performed on Day 1.  On Day 2, the subjects exercised for as long as possible while maintaining a minimum of 60 rpm (range 8.7-21.5 mins).  On Days 3, 4 the TTE was performed for an identical intensity and duration as Day 2.  For the TTE tests, similar cardiorespiratory variables as Day 1 were gathered, with the addition of a gastric balloon catheter and EMG. Hyperoxia trial.  On either Day 3 or 4, the TTE test was performed while inspiring humidified hyperoxic gas (FIO2 23-30%).  The level of hyperoxia was individually selected to reverse any hypoxaemia during the TTE, without raising arterial oxygen tension (PaO2) to unphysiological values (<150 mmHg).  Since changes in arterial blood gases throughout similar exercise protocols are reproducible (34, 109), we used measured PaO2 values from Day 2 to customize the hyperoxic inspirate for each subject.  During the hyperoxia trial, SaO2 was estimated using pulse oximeters.  Hyperoxic gas mixtures were made using a previously described system (39) that humidifies, stores, samples and delivers the inspired gas to the subject using large rigid pipes to minimize airflow resistance.  Proportional assist ventilator.  A proportional assist ventilator (PAV) was used to unload the respiratory muscles during one of the final TTE tests.  The PAV has been previously detailed (36) and is comparable to devices previously utilized by others (6, 15, 110).  Briefly, using a proportional solenoid valve and compressed air, positive airway  91  pressure was generated during inspiration in proportion to the subject’s respiratory effort, thereby unloading the respiratory muscles and reducing the WOB in a controlled manner. Electromyograms:  Surface EMG of the, rectus femoris, vastus lateralis and right and left costal diaphragm were recorded using surface electrodes (F-E15D160, Grass Technologies).  The electrodes were connected, with non-amplified leads (F-SL48 and F-P5IC3/REV1, Grass Technologies) to an amplifier (P5 Series, Grass Technologies).  Rectus femoris and Vastus lateralis electrodes were placed on the muscle bellies of the respective muscle.  The diaphragm electrodes were placed on the anterior axillary line in the sixth to eighth intercostal spaces.     The four sets of electrodes were placed in the same orientation as the muscle fibers and the ground electrode will be placed on the bony process of the anterior superior iliac spine or patella.  Peak-to-peak amplitudes and integrated area of the M-waves were measured for every twitch.  EMG signals were amplified (20x), band-pass filtered and the analog signals were A/D converted (PowerLab/16SP model ML 795, ADI, Colorado Springs, CO) and recorded simultaneously (10  kHz) using PowerLab data acquisition software (Chart v5.3, ADInstruments, Colorado Springs, CO). Quadriceps fatigue assessment:  Subjects had an inelastic strap wrapped around the malleoli on their right foot.  The strap was connected to a load cell (SML 1000, Interface, Scottsdale, AZ) via metal cable and carabiner.  The load cell was attached to a large wooden chair and was adjustable in three planes through customized hardware (80/20 Inc, East Columbia City, IN).  Adjustments were made to ensure traction along the load cell was parallel to the ground.  Care was taken to ensure the subject’s knee was in 90° flexion and the hip-knee-ankle alignment was similar between trials.  During the assessment of quadriceps fatigue, the participants were seated on the chair and secured via a seatbelt placed over their hips and across their shoulders.  Securing the subject was important to ensure only their quadriceps muscle group is activated during the maximal contractions and twitches.   92  Quadriceps fatigue was always assessed immediately before diaphragm fatigue.  To ensure the stimulator coil was in the same position we used both indelible ink and a 3tri-axial accelerometer (ADXL335, Adafruit) that securely fastened to the shaft of the stimulator coil.  The accelerometer outputted voltage signals that were in proportion to the 3-dimensional orientation of the device.  The signals were available in real-time to the investigators through the use of custom build software (LabView 8.0, National Instruments, Austin, TX).  Once satisfied with the initial coil placement, the orientation was marked in the software so that the location could be easily referenced for post-exercise twitch assessment.  Importantly, the accelerometer ensured the angle of the coil was identical between assessments.   Once the location of stimulation that elicited the greatest quadriceps twitch was found and marked, we performed a ramp protocol to ensure supramaximal twitches.  Briefly, we gathered 3 twitches, each separated by 30 secs, at power outputs corresponding to 60, 70, 80, 85, 90, 95 and 100% of the maximal power output (MagStim 200 Mono Pulse, MagStim, Whitland, Wales).  Thereafter, subjects performed a series of static maximal voluntary contractions (MVC).  Immediately after the contraction (~1 sec) a single twitch at 100% of stimulator output was performed.  Subjects performed a minimum of 6 MVC/twitches and the first two were disregarded because maximal potentiation was not achieved.  The series of potentiated twitches was performed before, immediately after (3-4 mins) and 10, 30 and 60 minutes after each TTE test.  The entire fatigue assessment including rigorous placement and the ramp protocol was performed on each TTE day (Days 2-4). Diaphragm Fatigue assessment:  In a subset of subjects (n=4, n=2 women), we assessed diaphragm fatigue via cervical magnetic stimulation.  Both phrenic nerves were stimulated using a high power 90-mm circular coil attached to the same magnetic stimulator used for quadriceps fatigue.  To find the site of optimal stimulation, we moved the coil between C5 and C7 while the subject was seated and their neck flexed.  Once the greatest  93  transdiaphragmtic twitch was obtained, the coil was traced in indelible ink to ensure similar placement.  As lung volume can influence twitch amplitude (73), all twitches were performed at end-expiratory lung volume.  Lung volumes were verified by examining end-expiratory oesophageal pressure prior to each stimulation.  Hereafter, the protocol for determining supramaximal twitch and the entire potentiated twitch protocol is identical to that described above for the quadriceps.   Arterial blood gases.  On Day 2, arterial blood samples were obtained from the radial artery as described previously (33).  Briefly, ~1.5 mL samples were drawn anaerobically into preheparanized syringes.  Samples were capped, any air evacuated and the blood was immediately analysed (< 2 mins) using a calibrated blood gas analyser (ABL80 CO-OX, Radiometer).  Blood gases, electrolytes and co-oximetry values were measured while bicarbonate was calculated.  The catheter was kept patent through the use of pressurized saline.  Arterial blood pressure was continuously monitored via a fluid-filled manometer located on an IV pole next to the subject, which was zeroed to the level of the heart.  Offline, blood gases were corrected for core temperature (114) as measured by a rapid response thermistor (Ret-1, Physitemp Instruments, Clinton, NJ, USA) placed in the oesophagus at a similar depth as the oesophageal balloon catheter. Data analysis.  Membrane excitability was inferred from the peak-to-peak amplitude and area of the m-waves for each potentiated twitch before and after exercise.  We determined the height and area of the m-wave for each contraction.  The amplitude of each quadriceps twitch was determined by subtracting the baseline force from the peak.  Raw EMG signals from the rectus femoris and vastus lateralis corresponding to each muscle contraction during each TTE trial were processed to determine muscle electrical activity.  Signals were band-pass filtered with a high and low cutoff frequency of 2000 and 30 Hz respectively and rectified.  Thereafter, customized software (Labview) detected the onset of  94  contraction in the vastus lateralis defined as a 2 standard deviation increase in baseline which lasted for 100 ms.  The area between each contraction was integrated and is expressed as a % of the 1st minute of each TTE (6).  The corresponding time between contractions of the vastus lateralis was used to mark where integration of the rectus femoris EMG was to begin.  We did not attempt to detect the onset of contraction time for the rectus femoris because in some subjects the contraction (to assist in hip flexion) occurred before the initiation of downward force.      The mechanical WOB was determined as previously detailed (40). For all presented data, the WOB was calculated using oesophageal pressure for pressure-volume loop integration and pressure-time product.  The method of assessing the WOB was selected because Campbell diagrams and using the transpulmonary pressure are not possible during assisted ventilation (i.e. PAV) (36).  All other cardiorespiratory variables were collected continuously using open-circuit spirometry as previously detailed (38).  Data presented in Table 8 is from the latter half of each TTE.  The latter 50% of each TTE was chosen because it represented a relative steady-state in V˙  O2 and a sustained elevated V˙  E > 50% of maximal V˙  E.   Repeat trials.  In some subjects (n=4, 2 women), we performed additional TTE tests (and associated fatigue measures) to ensure the reproducibility of our findings.  Specifically, we re-tested a control condition after the all experimental days in two subjects (n=1 male), performed secondary PAV days with similar (man) and different (woman) degrees of unloading.  None of the repeat trials are used for any subsequent analysis other than what is presented in the “Repeat trials” section of this manuscript.  Statistics.  Descriptive and maximal exercise test variables were compared using students unpaired t-tests.  A 2x3 (sex by TTE condition) repeated measures ANOVA was used to determine differences in cardiorespiratory variables for the TTE test.  For quadriceps  95  fatigue, a 2x3 (TTE condition by time) repeated measures ANOVA was used to determine difference in quadriceps fatigue.  When significant F ratios were detected, a Tukey post-hoc test was completed.  Pearson product moment correlation was used to determine the relationship between fraction of oxyhaemoglobin (FO2Hb) and attenuation in quadriceps fatigue. Significance was set at P<0.05 and data is presented as mean±SEM unless otherwise indicated.  5.3 Results Subjects. Subject descriptors and maximal exercise (Day 1) data are presented in Table 7.  Both groups were of similar age and had pulmonary function within normal limits.  For the initial maximal exercise test, men achieved a higher peak power, absolute and relative V˙  O2, but the sexes were of similar aerobic capacity as a % of predicted.  Despite men having a higher V˙  E, respiratory mechanics were similar between sexes; except the WOB was significantly greater in men.   96  Stimulator intensity (%max)60 70 80 90 100Twitch force (% maximal)020406080100ControlHyperoxiaPAVStimulator intensity (%max)60 70 80 90 100Twitch force (% maximal)020406080100Men WomenStimulator intensity (%max)60 70 80 90 100M-wave amplitude (%)020406080100Stimulator intensity (%max)60 70 80 90 100M-wave amplitude (%)020406080100Men WomenA BC D** ** *** ** Figure 18. Quadriceps twitch force (Panels A, B) and M-wave amplitude (Panel C, D) during magnetic stimulation of the femoral nerve at different stimulator outputs for men (Panel A, C) and women (Panel B, D). There was no difference between control, hyperoxia, or PAV for men or women at any stimulator intensity for twitch force or M-wave amplitude. PAV, proportional assist ventilator.  *, significantly different for all conditions from 100%. P<0.05.  97    Reproducibility of quadriceps neuromechanical variables.  Both sexes demonstrated a plateau in both quadriceps twitch force and m-wave amplitude with increasing stimulator intensity (Figure 18).  Furthermore, there were no differences between any of the experimental days (control, hyperoxia, PAV) at any stimulator intensity, regardless of sex or twitch/m-wave amplitude (P>0.05).  Men had a greater potentiated quadriceps twitch force and maximal voluntary contraction, but neither sex showed any difference between TTE trials for either variable (Table 8).  Between-day coefficient of variation for baseline potentiated twitch force was not different between the sexes (3.3±0.8 vs. 4.9±0.6% for men and women respectively; Range: men 1.3-8.5%, women 2.7-7.0%).    Arterial blood gases.  Arterial blood gases and oesophageal temperature throughout the control TTE are presented in Figure 19.  On average, subjects became hyperthermic (Panel A) and acidic (Panel C) in a time-dependent manner.  Although average PaO2 remained near rest, there was considerable variability.  Some subjects maintained their PaO2 within 5 mmHg for the duration of the TTE, whereas other had a considerable decrease.  Specifically, one male and one female had their PaO2 decrease up to 25-30 mmHg.  The two subjects who had the lowest PaO2 were the most aerobically fit; V˙  O2max 65 and 64 ml kg-1 min-1 for the man and women with lowest PaO2.  For both sexes, the major contributor to the change in oxyhaemoglobin saturation (>80%) was the result of temperature, pH and PCO2 related shifts in the oxygen dissociation curve, rather than changes PaO2.  However, both sexes (n=2 each sex) had subjects whose reduction in PaO2 was responsible for ~50% of the change in SaO2.  Table 9 displays the changes in blood gas variables from rest to exercise termination.  Men had a higher resting haemoglobin concentration and haematocrit, which resulted in greater CaO2.  Despite a lower FO2Hb at exercise termination, both groups became haemoconcentrated, resulting in a greater CaO2 at termination compared to rest  98  (Table 9).  However, if no desaturation was present, the increase in CaO2 would have been 0.7 ml dl-1 greater.  Both sexes demonstrated the anticipated changes in other variables, with no difference in the change from baseline between sexes (Table 9).   Figure 19. Group mean arterial blood gas and oesophageal temperature throughout the control (Day 2) time-to-exhaustion test. PaO2, arterial oxygen tension; FO2Hb, oxyhaemoglobin saturation. *, significantly different from baseline. P<0.05. 99  Table 8. Cardiorespiratory and quadriceps fatigue variables during the last 50% of the three TTE trials.  Control Hyperoxia PAV P value  M W M W M W Sex Trial Inter Cardiorespiratory          Workload (W kg-1) 3.78±0.2 3.44±0.2 3.79±0.2 3.44±0.2 3.79±0.2 3.44±0.2 NS NS NS HR (beats min-1) 179±3 181±3 175±4 180±4 173±4 181±3§ NS .018 .026 VT (l) 3.1±0.1 2.0±0.1* 3.2±0.2 2.1±0.1*ǂ 3.3±0.1 2.4±0.1*† <.001 <.001 NS fb (breaths min-1) 51±2 54±3 43±2 48±2† 47±2 48±2† NS <.001 NS V˙  E (l min-1) 158±7 108±4* 135±7 100±4*†ǂ 153±5 114±4* <.001 <.001 NS % max 85±2 91±3* 75±4 84±3* †ǂ 83±3 96±5* NS <.001 NS V˙  O2 (l min-1) 4.3±0.2 2.9±0.1* 4.1±0.1 2.9±0.1*ǂ 3.9±0.1 2.8±0.1*† <.001 <.001 .028 V˙  O2 (% control) - - 98±1 98±2ǂ 92±1 94±2 NS <.001 NS V˙  O2 (ml kg-1 min-1) 58±2 49±3* 56±1 49±3*ǂ 53±1 46±2*† .016 <.001 NS % max 96±1 94±1 94±2 92±2ǂ 88±1 88±2† NS <.001 NS V˙  CO2 (l min-1) 4.3±0.2 2.9±0.1* 4.1±0.1 2.9±0.1*ǂ 3.9±0.2 2.8±0.1*† <.001 <.001 .03 RER 1.00±0.02 1.00±0.01 1.02±.01 1.02±0.01 1.00±.01 1.02±.03 NS NS NS V˙  E/ V˙ O2 37±1 37±1 33±2 35±1†ǂ 39±1 42±2† NS <.001 NS V˙  E/ V˙ CO2 37±1 37±1 33±2 34±1†ǂ 39±1 41±1† NS <.001 NS Poeso swings(cmH2O) 48±2 41±3 39±2 37±2†ǂ 32±3 32±3† NS <.001 NS PTP (cmH2O l-1 sec-1) 568±28 499±36 459±13 456±27†ǂ 303±29 360±38† NS <.001 .004 ∆ PTP (% control) - - 84±4 92±5ǂ 54±4 70±4 <.001 <.001 <.001 FIO2(%) Room air 26.1±0.5 25.5±0.2 Room air    Quadriceps fatigue          Baseline twitch (N) 192±8 130±7* 189±9 133±6* 190±8 129±4* <.001 NS NS Range 158-234 113-174 160-230 108-157 155-229 116-149    Within series CV (%) 1.0±0.2 1.4±0.3 2.1±0.9 1.7±0.3 1.6±0.3 2.0±0.4 NS NS NS Baseline MVC (N) 640±21 430±27* 615±17 446±32* 618±21 456±34* <.001 NS NS Abbreviations: M, men; W, women; HR, heart rate; PetCO2, end-tidal carbon dioxide tension; VT, tidal volume; fb, breathing frequency; V˙  E, expired minute ventilation; V˙  O2, oxygen uptake; V˙  CO2, carbon dioxide output; RER, respiratory exchange ratio; ∆Poeso, oesophageal pressure swings; WOB, work of breathing; PTP, pressure-time product; FIO2, fraction of inspired oxygen; CV, coefficient of variation; MVC, maximal voluntary contraction. . *, significant main effect of sex; †, significantly different from control (both sexes pooled); ǂ, significantly different from PAV (both sexes pooled); §, significant interaction between sexes. P<0. 100  Time-to-exhaustion.  Cardiorespiratory variables for all TTE trials are presented in Table 8.  For both sexes compared to the control TTE, V˙  E was lower during the hyperoxia TTE and not different from the PAV TTE.  However, the PAV TTE resulted in a lower V˙  O2, despite identical workloads.  Despite men achieving a greater V˙  E, the oesophageal pressure swings and pressure time products were not different between the sexes, but were significantly lower during the PAV trial.    Quadriceps fatigue.  The percent change in quadriceps twitch force for both sexes is presented in Figure 20.  There were no significant differences in fatigue in any of the conditions between the sexes.  However, both the hyperoxia and PAV TTE resulted in significantly less quadriceps fatigue than the control trial.  The difference in quadriceps fatigue was present until 60 mins after test completion (Figure 20).  On average, inspiring hyperoxic gas resulted in a 31±5% attenuation in quadriceps muscle fatigue immediately post exercise.  However, the degree of attenuation ranged from ~-60% to ~0% and was significantly related to the nadir FO2Hb during the control TTE (Figure 21).  Performing the TTE on the PAV resulted in a lower WOB for both sexes (Figure 22, Panel A).  There was considerable variability for the decrease in WOB during the PAV trial and some subjects only had minimal changes in their WOB.  However, when the WOB is expressed as work unit per V˙  E, all subjects decreased in a similar manner (Figure 22, Panel B).  Overall, compared to the control TTE, the PAV TTE resulted in significantly less quadriceps fatigue (37±4% attenuation) and a lower V˙  O2, with no difference between sexes (Figure 22, Panel C).  However, men had a significantly greater reduction in their WOB compared to women (75±19 vs. 60±5% of control, P<0.05) (Figure 22, Panel C).  Compared to the control trial, there was no significant changes in V˙  E for either sex (P>0.05) (Panel C).    101  Table 9. Arterial blood gases variables at baseline and near exercise termination.  Baseline Termination Change from baseline  M W M W M W K+ (mmol l-1) 4.0±0.2 3.8±0.1 5.5±0.1† 5.4±0.1† 1.4±0.3 1.6±0.1 Ca2+ (mmol l-1) 1.19±0.01 1.17±.01 1.25±0.02† 1.26±0.01† 0.05±0.03 0.09±0.02 Na+ (mmol l-1) 142±0.1 142±5 145±0.9† 146±0.9† 3.1±0.9 3.8±0.8 Cl- (mmol l-1) 108±1 110±1 112±0.9† 112±0.9† 3.6±1.0 2.3±0.5 Hb (g dl-1) 14.5±0.3 12.9±0.2* 15.6±0.3† 14.1±0.2†* 1.2±0.3 1.1±0.2 Hct (%) 44±1 40±1* 48±1† 43±1†* 3.5±0.8 3.4±0.5 FHHb (%) 1.3±0.2 1.5±0.2 4.4±0.6† 4.2±0.6† 3.1±0.7 2.7±0.7 HCO3- (mmol l-1) 22.4±0.3 20.3±0.5* 12.1±0.9† 10.6±0.9†* -10.2±1.0  -9.6±1.4  PaO2 (mmHg) 99±4 100±1 94±3 96±2 -5±5 -3±3 [Range] [89-117] [93-104] [81-111] [86-106] [-23-21] [-17-8] PaCO2 (mmHg) [Range] 37±1 [29-41] 34±2 [26-38] 31±1† [24-34] 31±1† [28-33] -6±2  [-17-0]  -3±1  [-6-2] PAO2 (mmHg) [Range] 110±3 [100-124] 110±2 [106-120] 117±3† [113-123] 117±1† [113-120] 7±3  [-5-22] 7±1  [0-10] A-aDO2 (mmHg) [Range] 11±1 [5-15] 9±2 [7-17] 23±3† [12-33] 21±3† [8-34 12±3  [1-23] 11±3  [-1-25] CaO2 (ml dl-1) 20.1±0.5 18.1±0.2* 21.0±0.3† 18.9±0.4†* 0.9±0.4 0.7±0.2 1CaO2 (ml dl-1) 20.1±0.5 18.1±0.2* 21.7±0.4† 19.6±0.4†* 1.6±0.4 1.4±0.2 Values are mean±SEM. Abbreviations: M, men; W, women; Hb, haemoglobin; Hct, haematocrit; FHHb, fraction of deoxyhaemoglobin; PaCO2, arterial oxygen tension; PAO2, alveolar oxygen tension; A-aDO2, alveolar-to-arterial oxygen gradient; CaO2, arterial oxygen content; 1CaO2, ideal CaO2 if no desaturation was present. *, significantly different from men; †, significantly different from baseline. P<0.05.  Diaphragm fatigue (n=4).  Immediately after the control TTE, potentiated diaphragm twitch amplitude was 82±5% of baseline.  The PAV trial resulted in significantly less diaphragm fatigue (95±3% of control, P>0.05) for all subjects.  Baseline potentiated diaphragm twitches were not different between the control and PAV days (33.6±1.0 vs. 32.6±1.6 cmH2O, for the control and PAV trial respectively, P=0.65).  102   Figure 20. Changes in quadriceps twitch force across time and for the different conditions and each sex. PAV, proportional assist ventilator. *, significantly different than control day. P<0.05.  Repeat trials.  To verify the reproducibility of the trials we had a male and female subject complete an additional control trial 4-7 days after the final experimental days.  The differences between control trials are presented in Table 10.  A different male subject completed a second PAV trial 7 days after their first one with the attenuation in quadriceps fatigue being within 1% of each other.  Finally, another subject completed an additional PAV trial except the reduction in WOB was intentionally minimal (WOB >90% of control).  The lesser unloading resulted in the post exercise quadriceps twitch amplitude being similar to their control condition (31 vs 30% reduction for control and minimal unloading PAV, respectively).  Conversely, when the subject performed a PAV trial with maximal unloading (WOB <60% of control), their post exercise quadriceps twitch was 24% of the control condition.   103    Table 10. Repeated control TTE trials.  Subject A  (Male) Subject B Female) ∆ Baseline twitch (N) 3 8 ∆ 3 min post twitch (N) 2 7 ∆ V˙  O2 (l min-1) 0.05 0.09 ∆ V˙  O2 (ml kg-1 min-1) 0.7 0.1 ∆ V˙  E (l min-1) 1 6 Abbreviations: ∆ V˙  O2, change in oxygen uptake; ∆ V˙  E, change in ventilation.   5.4 Discussion Major findings.  We sought to determine the influence of EIAH and high respiratory muscle work on the development of locomotor fatigue in healthy men and women.  The findings from our study are three-fold.  First, regardless of sex, those who had the greatest decline in FO2Hb with exercise also showed the greatest degree of quadriceps fatigue attenuation when EIAH was experimentally prevented.   We interpret these observations to mean that the hypoxaemia that can accompany heavy exercise contributes to the development of quadriceps fatigue similarly between the sexes.  Second, we reduced the WOB to a greater degree in men yet both sexes showed similar level of quadriceps fatigue attenuation.  The similar fatigue attenuation with different degrees of unloading between the sexes suggests the high WOB associated with exercise contributes to locomotor muscle fatigue to a greater degree in women relative to men.  Third, during dynamic whole-body exercise, there does not appear to be a sex-difference in the fatigability of the quadriceps muscle group.  As such, the effect of any potential innate sex-difference in fatigue is likely minimal compared to the plethora of confounding factors elicited during whole-body dynamic exercise.  Overall, we have demonstrated that compared to men, quadriceps fatigue  104  in women is similarly exacerbated by EIAH, whereas the high WOB has a relatively greater effect.  Exercise-induced arterial hypoxaemia.  While all subjects demonstrated progressive hyperthermia and acidosis as exercise progressed, we observed significant variability with respect to blood gas homeostasis in both sexes.  Some subjects maintained PaO2 near resting values for the entire TTE, whereas others showed a consistent decline.  Furthermore, contrasting blood gases at baseline to exercise termination may not best represent the extent of EIAH, as we (33) and others (109) have demonstrated variable patterns of EIAH.  Some subjects had a progressive decline in PaO2, whereas others had an immediate drop that either returned to baseline or stayed depressed.  Nonetheless, regardless of sex, those who had the lowest FO2Hb demonstrated the most attenuation when breathing hyperoxic gas.  When breathing hyperoxia, oxyhaemoglobin saturation remained >98% for the duration of the TTE and the increased SaO2 would have raised CaO2.  As EIAH is reproducible between similar exercise trials (34), those who had the lowest FO2Hb during the control TTE, would have the greatest increase in CaO2 during the hyperoxia TTE.  It is important to note that CaO2 at exercise termination for the control TTE was not below baseline values.  Rather, due to an increase in haemoglobin and haematocrit, CaO2 rose progressively during exercise.  The relative haemoconcentration could be due to splenic erthyrocyte release (126) and is consistently observed during exercise (9, 122).  As such, the hyperoxia did not prevent a decline in CaO2, but rather, allowed the increase to be 50% greater (Table 9).  While the absolute increase in CaO2 is relatively modest, if we assume a similar O2 extraction, the greater CaO2 would increase mixed venous PO2 which is an important determinant of pulmonary gas exchange (133). We found no difference in the arterial blood gas variables between the sexes (Figure 19, Table 9).  We (33) and others (55) have found that women develop EIAH more readily  105  and to a more severe degree than men, but this is not a universal finding (64, 98).  We explain this apparent contradiction in several ways.  First, our current exercise modality was cycling, which is known to elicit a less severe decrease in PaO2, compared to running (64).  Second, the aforementioned studies investigating EIAH typically used either a V˙  O2max protocol or progressive stages.  Whereas, the current study utilized a TTE, which was designed to induce quadriceps muscle fatigue in part due to the sustained high work, acidosis and rise in core temperature, rather than progressively stress pulmonary gas exchange.  Finally, even within a relatively homogenous population, the appearance and severity of EIAH is variable (31) and the current study was not designed to address sex-differences in EIAH.    Nadir FO2Hb (%)0 90 91 92 93 94 95 96 97 98% Attenuation immediatly post exercise-80-60-40-200 MenWomenr2=0.79, p<0.0001 Figure 21. Relationship between the degree of quadriceps fatigue attenuation and the nadir oxyhaemoglobin saturation (FO2Hb) for the control trial. Quadriceps fatigue attenuation is the % change in potentiated twitch amplitude immediately after (3 mins) the hypoeroxia trial.  For all subjects, the nadir FO2Hb was the final blood samples taken immediately before exercise termination.  Regression line represents both  106  sexes pooled as each sex showed as significant relationship that was no different from each other.  SaO2, oxyhaemoglobin saturation.   Respiratory muscle work.  Lowering the mechanical WOB via the PAV resulted in a consistent attenuation of quadriceps fatigue (Figure 22).  The effect of lowered WOB on fatigue has been previously demonstrated in men (6, 110), but has not been shown in women.  We found that despite lowering the men’s WOB to a significantly greater degree, the attenuation in quadriceps fatigue was similar between the sexes (Figure 22).  The proposed mechanism behind quadriceps fatigue attenuation resulting from lowering the WOB involves sympathetically mediated redistribution of blood flow (54).  Specifically, in men, the high V˙  E during intense exercise results in a V˙  O2RM corresponding to ~10-12% of V˙  O2tot (3).  According to the Fick equation, the fraction of total cardiac output directed to an active tissue bed is proportional to the V˙  O2 of that tissue.  Thus, the respiratory musculature would command ~10-12% of cardiac output.  Since V˙  O2RM is linearly related to WOB (2, 38), any reduction in WOB results in a proportional change in V˙  O2RM.  The blood flow that was previously directed towards the respiratory musculature can now be re-directed to the active muscle tissue (54).  The increased blood flow would serve to lessen fatigue due to an increase in convective O2 delivery (5) and reduced metabolite accumulation (17, 62).  In women, the V˙  O2RM during intense exercise (V˙  E > 75% of max) represent a greater fraction of whole body V˙  O2RM (~15%) (38).  Therefore, despite the reduction in WOB being less in women than the men (75 vs 60% of control), the fraction of V˙  O2tot associated with the respiratory musculature was similar between the sexes for the PAV TTE.  Specifically, based on our V˙  O2RM/V˙  O2tot estimates at max exercise (38), a 25% and 40% reduction in the WOB for women and men would result in 3.5 and 3.8% of V˙  O2tot  no longer associated with the respiratory muscles.  Given the change in V˙  O2RM as a % of V˙  O2tot is similar between the sexes, it would follow that a comparable fraction of cardiac output will be redirected towards  107  the locomotor muscles.  A comparable increase in blood flow to the locomotor muscles would explain why the extent of fatigue attenuation was not different between the sexes.        Consistent with the reduction in WOB with the PAV is the decrease in whole body V˙  O2.  We and others (110) both found that whole body V˙  O2 decreased from 96% of V˙  O2max during the control trial to 88% for the PAV TTE.  The reduction in whole-body V˙  O2 principally represents the lesser WOB and therefore V˙  O2RM.  Critically, the change in V˙  O2 during the PAV trial was similar between the sexes (Table 8).  The similar change in V˙  O2 despite a different WOB attenuation provides further evidence  that a similar amount of cardiac output could be redirected resulting in similar fatigue attenuation.     During the hyperoxia TTE trial, there was a significant and consistent finding of a lower V˙  E which also resulted in a lower pressure-time product (Table 8).  The depressed V˙  E response with hyperoxia has been observed by others and is likely the result of decreased metabolite accumulation (109), as the increase in PaO2 with our modest increase in FIO2 would not be excessive (<150 mmHg) .  In contrast, changes in V˙  E during the PAV trial were variable and not statistically different from the control TTE for either sex.  In some subjects, there was a considerable increase in V˙  E during the PAV trial, resulting in only minor changes in the WOB when compared to the control (Figure 22A).  The relatively small change in WOB between control and PAV TTE is due to the curvilinear relationship between V˙  E and WOB (101).  When a subject generates a high V˙  E (>~80% of max) there is often an inflection point whereby any small change in V˙  E results in considerable increase in WOB.  However, when the WOB is expressed as work per liter of V˙  E, all subjects demonstrated a consistent decrease during the PAV trial (Figure 22B).  Specifically, the WOB unit cost of the increased V˙  E was less than what it would have been without the PAV.  Therefore, the modest decline in WOB during the PAV TTE (when subjects increased V˙  E) was still much lower than the WOB would have been without the PAV.    108   Figure 22. Work of breathing for the control trial and the proportional assist ventilation trial.  Regression lines in Panel A are redrawn from maximal exercise.  Small symbols in Panel A and B are individual data while large symbols are group averages.  In Panel C, “Fatigue” is the % difference in quadriceps fatigue between control and PAV trials. PAV, proportional assist ventilator; WOB, work of breathing; V˙  E, ventilation; V˙  O2, oxygen uptake. §, significantly different from control trial;  *, significantly different from control for both sexes; †, significantly different from women. P<0.05.  109  All the subjects who increased their V˙  E during the PAV trial would have expiratory flow limitation during their control TTE.  For example, the subject who had the largest increase in V˙  E during the PAV trial (+15 l min-1) had considerable expiratory flow limitation (>50% overlap of tidal flow-volume loops with MEFV) and a minimal hyperventilatory response (arterial carbon dioxide tension (PaCO2) -2 mmHg from baseline) during the control trial.  Unlike inspiring a helium mixture to lower the WOB and increase expiratory flow (13, 33, 139), the PAV does not permit expansion of the maximal expiratory flow-volume curve.  Therefore, the subject would have to increase their lung volume to access greater expiratory flows and produce the higher V˙  E.  Without the PAV, the consequence of the change in lung volume would be excessive WOB.  Specifically, as end-inspiratory lung volume increases, the lungs become relatively stiffer and the elastic WOB increases considerably (111).  Based on the example subject’s own V˙  E-WOB curve, if they sustained the elevated V˙  E without the PAV, their V˙  O2RM would have been >25% of total V˙  O2; which would be unsustainable.  Rather, the PAV allows for a V˙  E that would otherwise be energetically unfeasible.  The greater V˙  E would be beneficial because of a subsequently raised alveolar ventilation which, with other factors being similar, would raise PaO2 and lower PaCO2.  The raised PaO2 would increase SaO2 and the lower PaCO2 would increase pH; both of which would help to alleviate locomotor fatigue.  Mechanical ventilatory constraints limiting effective hyperpnea has been demonstrated previously in men (75) and women (87).  We have previously shown that when flow-limited subjects are given heliox, their V˙  E, PaO2 and subsequently CaO2 increases, whereas those who are not flow-limited show no change in V˙  E (33).  We observed a similar finding with regards to EFL in the current study, except the increase in V˙  E would have been mediated via reducing in WOB rather than expansion of the maximal expiratory flow-volume curve.  110   Sex difference in muscle fatigue.  We found no differences in the development of quadriceps muscle fatigue between the sexes after the control TTE.  Yet, others have found that women are typically more fatigue resistant and this finding persists even when matched for absolute strength (68).  The important difference in many of these studies relates to: the relative mass of the muscles, the location of the muscle and if the exercise is static or dynamic (65-67).  Typically the exercise paradigm where sex-difference are present involves small muscle mass and isometric contractions in the upper limb (113).  Whereas, when large (quadriceps) muscles perform dynamic contractions, there appears to be little difference between the sexes (66), which is consistent with our observation.  While we show no differences in fatigability between the sexes, we cannot fully exclude that there may still be innate sex-difference in muscle fatigue.  Rather, we suggest that during dynamic whole-body exercise there are many other factors known to influence the development of fatigue that likely outweigh any sex-specific influence.  For example, differences in training result in: changes in total force achievable which can compress microvasculature (69), altered muscle capillarization (112) and changes in muscle fiber type (140) ; all of which can influence fatigue.  Furthermore, during intense dynamic whole-body exercise there are many cardiorespiratory factors that are variable between subjects such as blood flow competition (21, 54), blood gas homeostasis (31) and difference in total WOB (37).  It is likely that the above confounding variables have a greater influence on fatigue development than any innate sex-differences.  Diaphragm fatigue.  After the TTE test on the PAV, we found no objective evidence of diaphragm fatigue.   Our findings of reduced diaphragm fatigue are similar to others (15) who also utilized a PAV to reduce the WOB during intense exercise.  Our findings emphasize the importance of work specific to the respiratory musculature, rather than locomotor muscles, in the development of diaphragm fatigue.  High respiratory muscle work  111  resulting in metabolite accumulation stimulates type III, IV afferents which causes increased sympathetic activity, blood flow redistribution and ultimately exacerbated locomotor fatigue (30).   The lack of diaphragm fatigue after the PAV trials further supports our hypothesis regarding blood flow redistribution causing fatigue attenuation.  However, we emphasize that not all subjects demonstrate diaphragm fatigue during exercise (52) and that fatigue per se is not necessary to elicit the respiratory muscle metaboreflex.  Rather, subjects must have a high degree of respiratory muscle work that is capable of producing metabolites (54, 108). Technical considerations.  Our principal finding is critically dependent on the ability to detect small changes in quadriceps fatigue.  Accordingly, we undertook several steps to ensure our results are representative of physiological changes rather than experimental variation.  First, prior to each TTE test, we were rigorous in our identification of the area which evoked the greatest twitch and performed a ramp protocol to confirm supramaximality.  Second, to ensure that the stimulator coil was placed on a similar position we outlined the coil placement in indelible ink, used an accelerometer to verify the coil placement and measured the coil distance from known anatomical part that were also marked in ink.  Given our within series (Table 8) and between day coefficient of variation (<5%) which was similar to others (109), we are confident our findings represent a physiological change. While a PAV permits some degree of customizability with respect to the degree of unloading, we were unable to reduce the WOB to a similar degree in all subjects.  On average, we were unable to unload women to a similar extent as men, however, there was overlap.  The variability in the degree of unloading arises from differences in subject: tolerance, mechanics and breathing patterns.  Notably for women, the relatively smaller tidal volume and greater frequency both decrease the total time available to unload.  Specifically, the PAV has an inherent static delay in the ability to generate positive mouth pressure.   112  Thus, when the reduced inspiratory time (greater breathing frequency) in women is coupled with a static delay, the fraction of a breath that is unloaded in women is less.  Finally,  manipulating intrathoracic pressure changes during exercise is known to induce heart-lung interactions, notably increases in pulmonary vascular resistance and a decrease in stroke volume (56).  Both aspects can lead to changes in cardiac output, which we were unable to assess in the present study.   Diaphragm fatigue was only measured in a subset (n=4) of subjects in order to confirm the absence of fatigue after the PAV TTE.  In a similar exercise paradigm to ours, others have demonstrated that exercise-induced diaphragm fatigue can be abolished when the WOB is reduced (15).  As such, our aim was to confirm that our iteration of a PAV was capable to eliminating diaphragm fatigue rather than investigating a sex-specific effect.   Conclusions.  Three conclusions can be drawn from our study.  First, although the extent and susceptibility of EIAH varies between (and within) the sexes, its implications for quadriceps muscle fatigue are not different.  Specifically, regardless of sex, those who develop the most severe EIAH demonstrate the most fatigue attenuation when EIAH is reversed.  Second, when compared to men, the reductions in WOB during exercise in women were significantly less, yet quadriceps fatigue was similarly attenuated.  We attribute the similar fatigue attenuation from unloading the WOB in women to a lesser extent to their greater relative V˙  O2RM.  Finally, during whole-body dynamic exercise with no experimental manipulations, the development of quadriceps muscle fatigue is similar between the sexes.  Overall, both EIAH and high WOB in healthy women can influence the development of quadriceps fatigue.  However, owing to their greater V˙  O2RM, alterations in WOB appears to influence quadriceps fatigue to a greater extent in women    113   Chapter Six: Conclusions 6.1 Overall summary High respiratory muscle work is known to influence the integrated response to dynamic whole body exercise.  Previously, these results were garnered from predominantly male subjects.  The work contained in this thesis highlights the similarity and differences with respect to the integrated response to high WOB in healthy men and women.  First, we found that the V˙  O2RM at a given V˙  E is greater in healthy women.  The greater V˙  O2RM at iso- V˙   E is the result of a greater WOB at similar V˙  E in women.  However, comparing the sexes at similar absolute values may not be optimal because of the effect of scaling.  For example, because absolute V˙  E is greater in men, the absolute V˙  O2RM at V˙  O2max is not different between the sexes.  Perhaps the most appropriate comparison is the relative V˙  O2RM at maximal exercise.  In this example, I found that at V˙  O2max, women dedicate a significantly greater fraction of total V˙  O2towards the respiratory muscles.  According to the Fick equation, the blood flow to an active tissue is proportional to its V˙  O2.  Therefore, it would surmise that women dedicate a greater fraction of cardiac output to their respiratory muscles.  However, this latter hypothesis requires rigorous testing. Second, I compared the influence of EIAH development of quadriceps fatigue in men and women.  I found that the influence of EIAH on quadriceps fatigue was not different between the sexes.  Specifically, regardless of sex, those who develop the most severe EIAH can show the greatest level of attenuation when the hypoxemia is experimentally reversed.  The rationale behind the fatigue attenuation when EIAH is reversed is the greater O2 delivery  114  from enhance CaO2.  While women may develop EIAH more severely and more often than men, this observation has not been confirmed in a large comparative study using optimal methods.  Therefore, determining the effect of EIAH on locomotor fatigue must be done on an individual basis because, regardless of sex, not all individuals develop EIAH. Third, it appears that the high WOB during exercise exacerbates quadriceps fatigue to a greater degree in women.  Specifically, the extent of quadriceps fatigue attenuation was similar between the sexes, yet, we reduced the WOB to a greater degree in men.  However, because women dedicate a greater fraction of V˙  O2 to their respiratory muscles, they require a less change in WOB in order to redistribute a similar amount of cardiac output.  That is, although the decrease in WOB was different between the sexes, our estimates of changes in leg blood flow (which are based on V˙  O2RM) are similar.  Therefore, the quadriceps muscles of both sexes would have received a similar amount of extra blood during the PAV TTE.  The similar extra blood flow, and subsequently O2 delivery, explains why the attenuation in quadriceps fatigue was not different.   6.2 Future directions There are several aspects regarding sex-differences in the integrated responses to exercise that remain unanswered.  Foremost, does our observation of greater relative oxygen cost of hyperpnea in healthy women translate into a greater fraction of total cardiac output being directed to their respiratory muscles?  Similarly, during intense exercise when the respiratory muscles are unloaded in men, there is a reflexive increase in active leg blood flow (54).  However, this redistribution of blood flow has not been demonstrated in women; and we hypothesize that the response would be enhanced in women.  Critical to the  115  respiratory related blood flow redistribution, would be the preservation of similar respiratory muscle metaboreflex in both sexes.  However, we know that at rest women are less responsive to vasocontrictor sympathetic activity due to an estrogen related upregulation of β2-adrenergic receptors (59) .  Yet, it is unknown is decreased sympathetic transduction is observed during exercise.  We observed no sex differences in in the severity of quadriceps fatigue after our exercise bouts.  Yet, others often demonstrate that women are more fatigue resistant than men(67).  Many studies that find women are more fatigue resistant involve relatively small muscle mass and static (rather than dynamic exercise).  Accordingly, further work is needed to assess possible sex-differences in fatigability with whole-body exercise.  Finally, there is very little data regarding the known heart-lung interactions that occur during respiratory muscle unloading.  Specifically, unloading is achieved by creating positive mouth pressure during inspiration.  In turn, the positive mouth pressure causes esophageal pressure to be relatively less negative.  A consequence is the possible reduction in venous return because of the diminished intrathoracic pressure swings (96); which is consistent with the lower stroke-volume observed with the PAV (56).  The limited information regarding heart-lung interactions with the PAV is exclusively based on men.  It is unknown if women will respond similarly, especially given the potential for sex-differences in cardiac function.   6.3 Conclusion Women, owing to their smaller airways, have a higher resistive and total WOB during exercise (117).  A consequence of this higher WOB is a greater relative and absolute V˙   116  O2RM in women.  The higher relative V˙  O2RM in women predisposed them to greater changes in locomotor fatigue from smaller changes in WOB.  Overall, the high WOB associated with intense exercise has a greater influence on the integrated response to whole-body exercise in women.             117  References  1. Aaker A, and Laughlin MH. Diaphragm arterioles are less responsive to α1- adrenergic constriction than gastrocnemius arterioles. Journal of Applied Physiology 92: 1808-1816, 2002. 2. Aaron EA, Johnson BD, Seow CK, and Dempsey JA. Oxygen cost of exercise hyperpnea: measurement. J Appl Physiol 72: 1810-1817, 1992. 3. Aaron EA, Seow KC, Johnson BD, and Dempsey JA. Oxygen cost of exercise hyperpnea: implications for performance. J Appl Physiol 72: 1818-1825, 1992. 4. Adams RP, and Welch HG. Oxygen uptake, acid-base status, and performance with varied inspired oxygen fractions. Journal of Applied Physiology 49: 863-868, 1980. 5. Amann M, and Calbet JAL. Convective oxygen transport and fatigue. Journal of Applied Physiology 104: 861-870, 2008. 6. Amann M, Pegelow DF, Jacques AJ, and Dempsey JA. Inspiratory muscle work in acute hypoxia influences locomotor muscle fatigue and exercise performance of healthy humans. Am J Physiol- Reg I Comp 293: R2036-R2045, 2007. 7. Amann M, Regan MS, Kobitary M, Eldridge MW, Boutellier U, Pegelow DF, and Dempsey JA. Impact of pulmonary system limitations on locomotor muscle fatigue in patients with COPD. Am J Physiol- Reg I Comp 299: R314-R324, 2010. 8. Andersen P, and Saltin B. Maximal perfusion of skeletal muscle in man. J Physiol 366: 233-249, 1985. 9. Astrand P, Cuddy T, Saltin B, and Stenberg J. Cardiac output during submaximal and maximal work. J Appl Physiol 19: 268-274, 1964. 10. Astrand P, and Rodahl K. Physiological bases of exercise. In: Textbook of work physiology. New York: McGraw-Hill, 1986. 11. ATS. Standardization of spirometry, 1994 update. American Thoracic Society. Am J Respir Crit Care Med 152: 1107-1136, 1995. 12. Babb TG. Exercise ventilatory limitation: The role of expiratory flow limitation. Exer Sport Sci Rev 41: 11-18, 2013. 13. Babb TG. Ventilation and respiratory mechanics during exercise in younger subjects breathing CO2 or HeO2. Respiration Physiology 109: 15-28, 1997. 14. Babb TG. Ventilatory response to exercise in subjects breathing CO2 or HeO2. Journal of Applied Physiology 82: 746-754, 1997. 15. Babcock MA, Pegelow DF, Harms CA, and Dempsey JA. Effects of respiratory muscle unloading on exercise-induced diaphragm fatigue. Journal of Applied Physiology 93: 201-206, 2002. 16. Babcock MA, Pegelow DF, McClaran SR, Suman OE, and Dempsey JA. Contribution of diaphragmatic power output to exercise-induced diaphragm fatigue. J Appl Physiol 78: 1710-1719, 1995. 17. Barclay JK. A delivery-independent blood flow effect on skeletal muscle fatigue. Journal of Applied Physiology 61: 1084-1090, 1986.  118  18. Bates JHT. Lung Mechanics: An Inverse Modeling Approach. Cambridge: Cambridge University Press, 2009. 19. Beidleman BA, Rock PB, Muza SR, Fulco CS, Forte VA, and Cymerman A. Exercise VË™e and physical performance at altitude are not affected by menstrual cycle phase. Journal of Applied Physiology 86: 1519-1526, 1999. 20. Cala SJ, Kenyon CM, Ferrigno G, Carnevali P, Aliverti A, Pedotti A, Macklem PT, and Rochester DF. Chest wall and lung volume estimation by optical reflectance motion analysis. Journal of Applied Physiology 81: 2680-2689, 1996. 21. Calbet JAL, Jensen-Urstad M, van Hall G, Holmberg H-C, Rosdahl H, and Saltin B. Maximal muscular vascular conductances during whole body upright exercise in humans. The Journal of Physiology 558: 319-331, 2004. 22. Campbell EJM. The Respiratory Muscles and the Mechanics of Breathing. Chicago: Year Book, 1958. 23. Coast JR, and Krause KM. Relationship of oxygen consumption and cardiac output to work of breathing. Med Sci Sport  Exer 25: 335-340, 1993. 24. Coast JR, Rasmussen SA, Krause KM, O'Kroy JA, Loy RA, and Rhodes J. Ventilatory work and oxygen consumption during exercise and hyperventilation. J Appl Physiol 74: 793-798, 1993. 25. Colebatch HJ, Greaves IA, and Ng KY. Exponential analysis of elastic recoil and aging in healthy males and females. J Appl Physiol 47: 683-691, 1979. 26. Dale HH, and Evans CL. Effects on the circulation of changes in the carbon-dioxide content of the blood. The Journal of Physiology 56: 125-145, 1922. 27. De Troyer A, and Boriek AM. Mechanics of the respiratory muscles. In: Comprehensive PhysiologyJohn Wiley & Sons, Inc., 2011. 28. Dempsey JA. Is the lung built for exercise? Med Sci Sports Exerc 18: 143-155, 1986. 29. Dempsey JA, Hanson PG, and Henderson KS. Exercise-induced arterial hypoxaemia in healthy human subjects at sea level. The Journal of Physiology 355: 161-175, 1984. 30. Dempsey JA, Romer L, Rodman J, Miller J, and Smith C. Consequences of exercise-induced respiratory muscle work. Respiratory Physiology & Neurobiology 151: 242-250, 2006. 31. Dempsey JA, and Wagner PD. Exercise-induced arterial hypoxemia. Journal of Applied Physiology 87: 1997-2006, 1999. 32. Derchak PA, Sheel AW, Morgan BJ, and Dempsey JA. Effects of expiratory muscle work on muscle sympathetic nerve activity. J Appl Physiol 92: 1539-1552, 2002. 33. Dominelli PB, Foster GE, Dominelli GS, Henderson WR, Koehle MS, McKenzie DC, and Sheel AW. Exercise-induced arterial hypoxaemia and the mechanics of breathing in healthy young women. J Physiol 591: 3017-3034, 2013. 34. Dominelli PB, Foster GE, Dominelli GS, Querido JS, Henderson WR, Koehle MS, and Sheel AW. Repeated exercise-induced arterial hypoxemia in a healthy untrained woman. Respiratory Physiology and Neurobiology 183: 201-205, 2012.  119  35. Dominelli PB, Guenette JA, Wilkie SS, Foster GE, and Sheel AW. Determinants of expiratory flow limitation in healthy women during exercise. Med Sci Sport  Exer 43: 1666-1674, 2011. 36. Dominelli PB, Henderson WR, and Sheel AW. A proportional assist ventilator to unload respiratory muscles experimentally during exercise in humans. Experimental Physiology 101: 754-767, 2016. 37. Dominelli PB, Molgat-Seon Y, Bingham D, Swartz PM, Road JD, Foster GE, and Sheel AW. Dysanapsis and the resistive work of breathing during exercise in healthy men and women. J Appl Physiol 119: 1105-1113, 2015. 38. Dominelli PB, Render JN, Molgat-Seon Y, Foster GE, Romer LM, and Sheel AW. Oxygen cost of exercise hyperpnoea is greater in women compared with men. The Journal of Physiology 593: 1965-1979, 2015. 39. Dominelli PB, Render JN, Molgat-Seon Y, Foster GE, and Sheel A. Precise mimicking of exercise hyperpnea to investigate the oxygen cost of breathing. Resp Physiol Neurobi 201: 14-23, 2014. 40. Dominelli PB, and Sheel AW. Experimental approaches to the study of the mechanics of breathing during exercise. Respiratory Physiology & Neurobiology 180: 147-161, 2012. 41. Dorrington KL, Balanos GM, Talbot NP, and Robbins PA. Extent to which pulmonary vascular responses to Pco2 and Po2 play a functional role within the healthy human lung. Journal of Applied Physiology 108: 1084-1096, 2010. 42. Duhamel TA, Green HJ, Sandiford SD, Perco JG, and Ouyang J. Effects of progressive exercise and hypoxia on human muscle sarcoplasmic reticulum function. Journal of Applied Physiology 97: 188-196, 2004. 43. Eckermann P, and Millahn HP. Der Sauerstoffverbrauch der Atmungsmuskulatur bei Frauen. Int Z Angew Physiol 19: 168-172, 1962. 44. Eves ND, Petersen SR, Haykowsky MJ, Wong EY, and Jones RL. Helium-Hyperoxia, Exercise, and Respiratory Mechanics in Chronic Obstructive Pulmonary Disease. American Journal of Respiratory and Critical Care Medicine 174: 763-771, 2006. 45. Foster GE, Koehle MS, Dominelli PB, Mwangi FM, Onywera VO, Boit MK, Tremblay JC, Boit C, and Sheel AW. Pulmonary Mechanics and Gas Exchange during Exercise in Kenyan Distance Runners. Medicine and Science in Sports and Exercise 46: 702-710, 2014. 46. Gallagher CG, and Younes M. Effect of pressure assist on ventilation and respiratory mechanics in heavy exercise. Journal of Applied Physiology 66: 1824-1837, 1989. 47. Goldman MD, Grimby G, and Mead J. Mechanical work of breathing derived from rib cage and abdominal V-P partitioning. J Appl Physiol 41: 752-763, 1976. 48. Green M, Mead J, and Turner JM. Variability of maximum expiratory flow-volume curves. J Appl Physiol 37: 67-74, 1974. 49. Grimby G, Bunn J, and Mead J. Relative contribution of rib cage and abdomen to ventilation during exercise. J Appl Physiol 24: 159-166, 1968.  120  50. Guenette JA, Dominelli PB, Reeve SS, Durkin CM, Eves ND, and Sheel AW. Effect of thoracic gas compression and bronchodilation on the assessment of expiratory flow limitation during exercise in healthy humans. Resp Physiol Neurobi 170: 279-286, 2010. 51. Guenette JA, Querido JS, Eves ND, Chua R, and Sheel AW. Sex differences in the resistive and elastic work of breathing during exercise in endurance-trained athletes. Am J Physiol- Reg I 297: R166-R175, 2009. 52. Guenette JA, Romer LM, Querido JS, Chua R, Eves ND, Road JD, McKenzie DC, and Sheel AW. Sex differences in exercise-induced diaphragmatic fatigue in endurance-trained athletes. Journal of Applied Physiology 109: 35-46, 2010. 53. Guenette JA, Witt JD, McKenzie DC, Road JD, and Sheel AW. Respiratory mechanics during exercise in endurance-trained men and women. The Journal of Physiology 581: 1309-1322, 2007. 54. Harms CA, Babcock MA, McClaran SR, Pegelow DF, Nickele GA, Nelson WB, and Dempsey JA. Respiratory muscle work compromises leg blood flow during maximal exercise. J Appl Physiol 82: 1573-1583, 1997. 55. Harms CA, McClaran SR, Nickele GA, Pegelow DF, Nelson WB, and Dempsey JA. Exercise-induced arterial hypoxaemia in healthy young women. J Physiol 507: 619-628, 1998. 56. Harms CA, Wetter TJ, McClaran SR, Pegelow DF, Nickele GA, Nelson WB, Hanson P, and Dempsey JA. Effects of respiratory muscle work on cardiac output and its distribution during maximal exercise. J Appl Physiol 85: 609-618, 1998. 57. Harms CA, Wetter TJ, St. Croix CM, Pegelow DF, and Dempsey JA. Effects of respiratory muscle work on exercise performance. J Appl Physiol 89: 131-138, 2000. 58. Hart E, and Charkoudian N. Sympathetic Neural Regulation of Blood Pressure: Influences of Sex and Aging. Physiology 29: 8-15, 2014. 59. Hart EC, Charkoudian N, Wallin BG, Curry TB, Eisenach J, and Joyner MJ. Sex and ageing differences in resting arterial pressure regulation: the role of the B-adrenergic receptors. J Physiol 589: 5285-5297, 2011. 60. Hicks AL, Kent-Braun J, and Ditor DS. Sex differences in human skeletal muscle fatigue. Exer Sport Sci Rev 29: 109-112, 2001. 61. Hogan MC, Cox RH, and Welch HG. Lactate accumulation during incremental exercise with varied inspired oxygen fractions. Journal of Applied Physiology 55: 1134-1140, 1983. 62. Hogan MC, Gladden LB, Grassi B, Stary CM, and Samaja M. Bioenergetics of contracting skeletal muscle after partial reduction of blood flow. Journal of Applied Physiology 84: 1882-1888, 1998. 63. Hogan MC, Richardson RS, and Haseler LJ. Human muscle performance and PCr hydrolysis with varied inspired oxygen fractions: a 31P-MRS study. Journal of Applied Physiology 86: 1367-1373, 1999. 64. Hopkins SR, Barker RC, Brutsaert TD, Gavin TP, Entin P, Olfert IM, Veisel S, and Wagner PD. Pulmonary gas exchange during exercise in women: effects of exercise type and work increment. Journal of Applied Physiology 89: 721-730, 2000.  121  65. Hunter SK. Sex Differences and Mechanisms of Task-Specific Muscle Fatigue. Exercise and Sport Sciences Reviews 37: 113-122, 2009. 66. Hunter SK. Sex differences in fatigability of dynamic contractions. Experimental Physiology 101: 250-255, 2016. 67. Hunter SK. Sex differences in human fatigability: mechanisms and insight to physiological responses. Acta Physiologica 210: 768-789, 2014. 68. Hunter SK, Critchlow A, Shin I-S, and Enoka RM. Men are more fatigable than strength-matched women when performing intermittent submaximal contractions. Journal of Applied Physiology 96: 2125-2132, 2004. 69. Hunter SK, and Enoka RM. Sex differences in the fatigability of arm muscles depends on absolute force during isometric contractions. Journal of Applied Physiology 91: 2686-2694, 2001. 70. Hyatt RE. Expiratory flow limitation. J Appl Physiol 55: 1-7, 1983. 71. Iandelli I, Aliverti A, Kayser B, Dellaca R, Cala SJ, Duranti R, Kelly S, Scano G, Sliwinski P, Yan S, Macklem PT, and Pedotti A. Determinants of exercise performance in normal men with externally imposed expiratory flow limitation. J Appl Physiol 92: 1943-1952, 2002. 72. Janicki JS, Sheriff DD, Robotham JL, and Wise RA. Cardiac Output During Exercise: Contributions of the Cardiac, Circulatory, and Respiratory Systems. In: Comprehensive PhysiologyJohn Wiley & Sons, Inc., 2011, p. 649-704. 73. Johnson BD, Babcock MA, Suman OE, and Dempsey JA. Exercise-induced diaphragmatic fatigue in healthy humans. J Physiol 460: 385-405, 1993. 74. Johnson BD, Badr M, and Dempsey JA. Impact of the aging pulmonary system on the response to exercise. Clinics in chest medicine 15: 229, 1994. 75. Johnson BD, Saupe KW, and Dempsey JA. Mechanical constraints on exercise hyperpnea in endurance athletes. J Appl Physiol 73: 874-886, 1992. 76. Johnson BD, Weisman IM, Zeballos RJ, and Beck KC. Emerging Concepts in the Evaluation of Ventilatory Limitation During Exercise. Chest 116: 488-503, 1999. 77. Klas J, and Dempsey JA. Voluntary versus reflex regulation of maximal exercise flow: volume loops. Am Rev Respir Dis 139: 150-156, 1989. 78. Kregenow DA, and Swenson ER. The lung and carbon dioxide: implications for permissive and therapeutic hypercapnia. European Respiratory Journal 20: 6-11, 2002. 79. Laghi F, D'Alfonso N, and Tobin MJ. Pattern of recovery from diaphragmatic fatigue over 24 hours. J Appl Physiol 79: 539-546, 1995. 80. Livermore N, Dimitri A, Sharpe L, McKenzie DK, Gandevia SC, and Butler JE. Cognitive behaviour therapy reduces dyspnoea ratings in patients with chronic obstructive pulmonary disease (COPD). Resp Physiol Neurobi 216: 35-42, 2015. 81. Lorenzo S, and Babb TG. Oxygen cost of breathing and breathlessness during exercise in nonobese women and men. Med Sci Sport Exer 44: 1043-1048 2012. 82. Lua AC, Shi KC, and Chua LP. Proportional assist ventilation system based on proportional solenoid valve control. Medical Engineering & Physics 23: 381-389, 2001. 83. Lublin A, Wolfenson D, and Berman A. Sex differences in blood flow distribution of normothermic and heat-stressed rabbits. Am J Physiol- Reg I 268: R66-R71, 1995.  122  84. MacNutt MJ, De Souza MJ, Tomczak SE, Homer JL, and Sheel AW. Resting and exercise ventilatory chemosensitivity across the menstrual cycle. J Appl Physiol 112: 737-747, 2012. 85. Manohar M. Inspiratory and expiratory muscle perfusion in maximally exercised ponies. J Appl Physiol 68: 544-548, 1990. 86. Martin TR, Castile RG, Fredberg JJ, Wohl ME, and Mead J. Airway size is related to sex but not lung size in normal adults. J Appl Physiol 63: 2042-2047, 1987. 87. McClaran SR, Harms CA, Pegelow DF, and Dempsey JA. Smaller lungs in women affect exercise hyperpnea. J Appl Physiol 84: 1872-1881, 1998. 88. McCool FD, McCann DR, Leith DE, and Hoppin FG. Pressure-flow effects on endurance of inspiratory muscles. J Appl Physiol 60: 299-303, 1986. 89. McCool FD, Tzelepis GE, Leith DE, and Hoppin FG. Oxygen cost of breathing during fatiguing inspiratory resistive loads. J Appl Physiol 66: 2045-2055, 1989. 90. Mead J. Dysanapsis in normal lungs assessed by the relationship between maximal flow, static recoil, and vital capacity. Am Rev Respir Dis 121: 339-342, 1980. 91. Mead J. Measurement of Inertia of the Lungs at Increased Ambient Pressure. Journal of Applied Physiology 9: 208-212, 1956. 92. Mead J, Turner JM, Macklem PT, and Little JB. Significance of the relationship between lung recoil and maximum expiratory flow. J Appl Physiol 22: 95-108, 1967. 93. Milic-Emili J, Mead J, Turner JM, and Glauser EM. Improved technique for estimating pleural pressure from esophageal balloons. J Appl Physiol 19: 207-211, 1964. 94. Miller AEJ, MacDougall J, Tarnopolsky M, and Sale D. Gender differences in strength and muscle fiber characteristics. Eur J Appl Physiol O 66: 254-262, 1993. 95. Miller JD, Hemauer SJ, Smith CA, Stickland MK, and Dempsey JA. Expiratory threshold loading impairs cardiovascular function in health and chronic heart failure during submaximal exercise. J Appl Physiol 101: 213-227, 2006. 96. Miller JD, Pegelow DF, Jacques AJ, and Dempsey JA. Skeletal muscle pump versus respiratory muscle pump: modulation of venous return from the locomotor limb in humans. The Journal of Physiology 563: 925-943, 2005. 97. Nielsen M. Die Respirationsarbeit bei Korperruhe und bei Muskl arbeit. Archiv Fur Physiologie 74: 299-316, 1936. 98. Olfert IM, Balouch J, Kleinsasser A, Knapp A, Wagner H, Wagner PD, and Hopkins SR. Does gender affect human pulmonary gas exchange during exercise? The Journal of Physiology 557: 529-541, 2004. 99. Olsen CO, Tyson GS, Maier GW, Davis JW, and Rankin JS. Diminished stroke volume during inspiration: a reverse thoracic pump. Circulation 72: 668-679, 1985. 100. Olson TP, Joyner MJ, Dietz NM, Eisenach JH, Curry TB, and Johnson BD. Effects of respiratory muscle work on blood flow distribution during exercise in heart failure. The Journal of Physiology 588: 2487-2501, 2010. 101. Otis AB. The work of breathing. Physiol Rev 34: 449-458, 1954. 102. Otis AB. The work of breathing. Washington, DC: American Physiological Society, 1964.  123  103. Otis AB, Fenn WO, and Rahn H. Mechanics of Breathing in Man. J Appl Physiol 2: 592-607, 1950. 104. Pellegrino R, Brusasco V, Rodarte JR, and Babb TG. Expiratory flow limitation and regulation of end-expiratory lung volume during exercise. J Appl Physiol 74: 2552-2558, 1993. 105. Poole DC, Gaesser GA, Hogan MC, Knight DR, and Wagner PD. Pulmonary and leg VO2 during submaximal exercise: implications for muscular efficiency. J Appl Physiol 72: 805-810, 1992. 106. Ravussin E, Lillioja S, Anderson TE, Christin L, and Bogardus C. Determinants of 24-hour energy expenditure in man. Methods and results using a respiratory chamber. The Journal of Clinical Investigation 78: 1568-1578, 1986. 107. Renggli AS, Verges S, Notter DA, and Spengler CM. Development of respiratory muscle contractile fatigue in the course of hyperpnoea. Resp Physiol Neurobi 164: 366-372, 2008. 108. Rodman JR, Henderson KS, Smith CA, and Dempsey JA. Cardiovascular effects of the respiratory muscle metaboreflexes in dogs: rest and exercise. Journal of Applied Physiology 95: 1159-1169, 2003. 109. Romer LM, Haverkamp HC, Lovering AT, Pegelow DF, and Dempsey JA. Effect of exercise-induced arterial hypoxemia on quadriceps muscle fatigue in healthy humans. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 290: R365-R375, 2006. 110. Romer LM, Lovering AT, Haverkamp HC, Pegelow DF, and Dempsey JA. Effect of inspiratory muscle work on peripheral fatigue of locomotor muscles in healthy humans. The Journal of Physiology 571: 425-439, 2006. 111. Roussos C, and Campbell EJM editors. Respiratory muscle energetics. Bethesda, MD: 1986, p. 481-509. 112. Saltin B, Blomqvist G, Mitchell JH, Johnson RL, Wildenthal K, Chapman CB, Frenkel E, Norton W, Siperstein M, Suki W, Vastagh G, and Prengler A. A Longitudinal Study of Adaptive Changes in Oxygen Transport and Body Composition. Circulation 38: VII-1-VII-78, 1968. 113. Senefeld J, Yoon T, Bement MH, and Hunter SK. Fatigue and recovery from dynamic contractions in men and women differ for arm and leg muscles. Muscle & Nerve 48: 436-439, 2013. 114. Severinghaus JW. Blood gas calculator. Journal of Applied Physiology 21: 1108-1116, 1966. 115. Sharma P, Morris NR, and Adams L. Effect of Experimental Modulation of Mood on Perception of Exertional Dyspnea in Healthy Subjects. Journal of Applied Physiology 2015. 116. Sheel AW, Derchak PA, Morgan BJ, Pegelow DF, Jacques AJ, and Dempsey JA. Fatiguing inspiratory muscle work causes reflex reduction in resting leg blood flow in humans. J Physiol 537: 277-289, 2001. 117. Sheel AW, Dominelli PB, and Molgat-Seon Y. Revisiting dysanapsis: sex-based differences in airways and the mechanics of breathing during exercise. Experimental Physiology 101: 213-218, 2016.  124  118. Sheel AW, and Guenette JA. Mechanics of Breathing during Exercise in Men and Women: Sex versus Body Size Differences? Exercise and Sport Sciences Reviews 36: 128-134 110.1097/JES.1090b1013e31817be31817f31810, 2008. 119. Sheel AW, and Guenette JA. Mechanics of breathing during exercise in men and women: sex versus body size differences? Exercise and sport sciences reviews 36: 128-134, 2008. 120. Sheel AW, Guenette JA, Yuan R, Holy L, Mayo JR, McWilliams AM, Lam S, and Coxson HO. Evidence for dysanapsis using computed tomographic imaging of the airways in older ex-smokers. J Appl Physiol 107: 1622-1628, 2009. 121. Shephard RJ. The oxygen cost of breathing during vigorous exercise. Quart J Exper Physiol 51: 336-350, 1966. 122. Sjogaard G, and Saltin B. Extra- and intracellular water spaces in muscles of man at rest and with dynamic exercise. Am J Physiol- Reg I 12: R271-R280, 1982. 123. Smith JR, Rosenkranz SK, and Harms CA. Dysanapsis ratio as a predictor for expiratory flow limitation. Resp Physiol Neurobi 198: 25-31, 2014. 124. St Croix CM, Morgan BJ, Wetter TJ, and Dempsey JA. Fatiguing inspiratory muscle work causes reflex sympathetic activation in humans. J Physiol 529: 493-504, 2000. 125. Stark-Leyva KN, Beck KC, and Johnson BD. Influence of expiratory loading and hyperinflation on cardiac output during exercise. Journal of Applied Physiology 96: 1920-1927, 2004. 126. Stewart IB, Warburton DER, Hodges ANH, Lyster DM, and McKenzie DC. Cardiovascular and splenic responses to exercise in humans. Journal of Applied Physiology 94: 1619-1626, 2003. 127. Tan W, Bourbeau J, Hernandez P, Chapman K, Cowie R, FitzGerald M, Aaron S, Marciniuk D, Maltais F, O'Donnell D, Goldstein R, Sin D, Chan-Yeung M, Manfreda J, Anthonisen N, Tate R, Sears M, Siersted H, Becklake M, Ernst P, Bowie D, Sweet L, and Til LV. Canadian prediction equations of spirometric lung function for Caucasian adults 20 to 90 years of age: Results from the Canadian Obstructive Lung Disease (COLD) study and the Lung Health Canadian Environment (LHCE) study. Canadian Respiratory Journal 18: 321 - 326, 2011. 128. Taylor BJ, How SC, and Romer LM. Exercise-induced abdominal muscle fatigue in healthy humans. J Appl Physiol 100: 1554-1562, 2006. 129. Taylor BJ, and Romer LM. Effect of expiratory muscle fatigue on exercise tolerance and locomotor muscle fatigue in healthy humans. J Appl Physiol 104: 1442-1451, 2008. 130. Thurlbeck WM. Postnatal human lung growth. Thorax 37: 564-571, 1982. 131. Topin N, Mucci P, Hayot M, Prefaut C, and Ramonatxo M. Gender influence on the oxygen consumption of the respiratory muscles in young and older healthy individuals. Intl J Sports Med 24: 559-564, 2003. 132. von Leupoldt A, and Dahme B. Psychological aspects in the perception of dyspnea in obstructive pulmonary diseases. Respiratory Medicine 101: 411-422, 2007. 133. Wagner PD. Influence of mixed venous PO2 on diffusion of O2 across the pulmonary blood: gas barrier. Clinical Physiology 2: 105-115, 1982.  125  134. Wanke T, Formanek D, Schenz G, Popp W, Gatol H, and Zwick H. Mechanical load on the ventilatory muscles during an incremental cycle ergometer test. Eur Respir J 4: 385-392, 1991. 135. Wasserman K, Hansen JE, and Sue D. Principles of Exercise Testing and Interpretations. Baltimore: Lippincott Williams & Wilkins, 1999. 136. Wetter TJ, Harms CA, Nelson WB, Pegelow DF, and Dempsey JA. Influence of respiratory muscle work onVË™o 2 and leg blood flow during submaximal exercise. Journal of Applied Physiology 87: 643-651, 1999. 137. Whipp BJ, and Pardy RL. Breathing During Exercise. In: Comprehensive PhysiologyJohn Wiley & Sons, Inc., 2011. 138. Whittenberger JL, McGregor M, Berglund E, and Borst HG. Influence of state of inflation of the lung on pulmonary vascular resistance. Journal of Applied Physiology 15: 878-882, 1960. 139. Wilkie SS, Dominelli PB, Sporer BC, Koehle MS, and Sheel AW. Heliox breathing equally influences respiratory mechanics and cycling performance in trained males and females. Journal of Applied Physiology 118: 255-264, 2015. 140. Yan Z, Okutsu M, Akhtar YN, and Lira VA. Regulation of exercise-induced fiber type transformation, mitochondrial biogenesis, and angiogenesis in skeletal muscle. Journal of Applied Physiology 110: 264-274, 2011. 141. Younes M. Proportional Assist Ventilation, a New Approach to Ventilatory Support: Theory. American Review of Respiratory Disease 145: 114-120, 1992. 142. Younes M, Bilan D, Jung D, and Kroker H. An apparatus for altering the mechanical load of the respiratory system. Journal of Applied Physiology 62: 2491-2499, 1987. 143. Younes M, and Kivinen G. Respiratory mechanics and breathing pattern during and following maximal exercise. J Appl Physiol 57: 1773-1782, 1984. 144. Younes M, Puddy A, Roberts D, Light RB, Quesada A, Taylor K, Oppenheimer L, and Cramp H. Proportional Assist Ventilation: Results of an Initial Clinical Trial. American Review of Respiratory Disease 145: 121-129, 1992.   

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0340564/manifest

Comment

Related Items