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Exercise-induced bronchoconstriction – the effect of inhaled ß₂-agonists on athletic performance in… Koch, Sarah 2017

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EXERCISE-INDUCED BRONCHOCONSTRICTION – THE EFFECTS OF INHALED ß2-AGONISTS ON ATHLETIC PERFORMANCE  IN HUMANS by  Sarah Koch  Diplom, University of Potsdam, 2009 MSc., The University of British Columbia, 2011  A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT 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 2017 © Sarah Koch, 2017 ii  Abstract The purpose of this thesis was to investigate the ergogenic potential of inhaled ß2-agonists (IBAs) in athletes with and without exercise-induced bronchoconstriction (EIB) from multiple angles: by assessing the effect of 400 µg of salbutamol, a commonly used IBA, in female athletes (Chapter 2); and by analyzing athletic performance after the inhalation of the maximal daily dose of salbutamol, i.e. 1600 µg, by taking dose per kg of body mass into consideration (Chapter 3). Furthermore, four methods to calculate the fall index (FI) in the eucapnic voluntary hyperpnea (EVH) challenge were evaluated (Chapter 4).   Female (Chapter 2) and male (Chapter 3) cyclists were screened for EIB using the EVH challenge. On two subsequent visits, athletes performed one 10-km time trial per visit. In a randomized order, athletes inhaled either salbutamol or placebo before completing the time trials. Athletic performance was assessed using mean power output. In Chapter 4, the FI of the EVH challenges from Chapters 2 and 3 were calculated using four methods and compared to the pulmonary response to the placebo time trial. Additionally, the intensity of the EVH target ventilation, calculated as 30 x forced expiratory volume in 1 second (FEV1), was evaluated by comparing it to the highest generated ventilations in a graded maximal exercise test (GXT).   Despite significant increases in FEV1 after IBA use, athletic performance was not improved in female (Chapter 2) or male (Chapter 3) athletes, regardless of EVH status. In women, power output was decreased, despite a significant increase in oxygen consumption, suggestive of an increased stimulation of women’s adrenergic β2-system. Similarly, in male athletes, significant increases in heart rate, ventilation, and leg discomfort could indicate an upregulation of the iii  adrenergic nervous system, independent of relative IBA dose. Lastly, there were significant differences between the four FI calculations (Chapter 4), influencing the EVH interpretations. Normalizing FIs by the ventilations achieved during voluntary hyperpnea was not supported in athletes with high percent predicted values for FEV1 due to an increased risk of false-positives. The intensity of the EVH target ventilation ranged between 67% - 135% when normalized to the GXT ventilations.   iv  Lay summary Up to 8% of Olympians suffer from the temporary narrowing of the airways following exercise, called exercise-induced bronchoconstriction (EIB). I investigated whether the inhalation of salbutamol, a commonly used airway-widening medication, leads to increases in athletic performance in female (Chapter 2) and male athletes (Chapter 3) with and without EIB. I also evaluated four methods to determine the decrease in lung function in response to a diagnostic test for EIB called the Eucapnic Voluntary Hyperpnea (EVH) challenge (Chapter 4). Female and male athletes did not improve athletic performance after the inhalation of salbutamol when compared to placebo. This finding was independent of EIB diagnosis. Analyzing the decrease in lung function in response to the EVH challenge with four different methods led to significant differences, affecting the EIB diagnoses based on the EVH challenge. This suggets that the method used should be chosen with care.   v  Preface This thesis is formatted as a manuscript-based thesis, as required by the Graduate and Postdoctoral Studies’ guidelines. Chapters 2 and 3 have been published, and copyrights for both manuscripts have been obtained from the publishers of the Journal of Science and Medicine in Sport (Chapter 2) and Medicine & Science in Sports & Exercise (Chapter 3). By agreeing to the publishers’ conditions, manuscripts were not permitted to be rewritten; however, minimal adaptions to serve this thesis were permitted. Unfortunately, this leads to some repetition, especially in the introduction sections of the chapters.   The literature review outlined in Chapter 1 was drafted by me and reviewed by my supervisor and committee members, Dr. Koehle, Dr. Guenette, and Dr. Carlsten.  A version of Chapter 2 has been published as Koch S., Karacabeyli D., Galts C., MacInnis MJ., Sporer BC., and Koehle MS. (2015) Effects of inhaled bronchodilators on lung function and cycling performance in female athletes with and without exercise-induced bronchoconstriction. Journal of Science and Medicine in Sport. 18 (5):607-612. Drs. Koehle, Sporer, and MacInnis assisted me in the study design, data analysis, and manuscript editing. I recruited all study participants, conducted all study visits, performed the data analysis, and wrote most of the manuscript. Mr. Karacabeyli and Mr. Galts assisted in the collection of the data.  The study presented in Chapter 2 is a continuation of my Masters of Science (MSc) work. In my MSc project, I investigated the effect of IBAs on athletic performance in male athletes. The primary purpose of Chapter 2 was to assess the ergogenic potential of IBAs in women, a study population that had been overlooked previously. A secondary purpose of Chapter 2 was to vi  investigate sex differences in the response to IBAs on lung function and athletic performance. Using an identical study design, I compared the spirometric and athletic performance parameters collected in female athletes during my doctoral work to the data I assessed during my MSc training in male athletes. In Chapter 2, I present data collected and analyzed during my doctoral work on trained female athletes, including new analyses to address sex differences in the response to IBAs on lung function and athletic performance. Data on male athletes from my MSc work, which were used for the sex comparisons, are not presented in this doctoral thesis but can be found published as Koch S., MacInnis MJ., Sporer BC., Rupert JL., and Koehle MS. (2015) Inhaled salbutamol does not affect athletic performance in asthmatic and non-asthmatic cyclists. British Journal of Sport Medicine. 49 (1):51-55. Additionally, an electronic copy of the complete MSc project can be accessed on cIRcle, the University of British Columbia’s digital repository for research and teaching materials at:  https://open.library.ubc.ca/cIRcle/collections/ubctheses/24/items/1.0072287.  A version of Chapter 3 has been published as Koch S., Ahn JR., and Koehle MS. (2015) High-dose inhaled salbutamol does not improve 10-km cycling time trial performance. Medicine & Science in Sports & Exercise. 47(11):2373-2379. Dr. Koehle secured funding and provided supervision and guidance in the study design, data analysis, and manuscript preparation. I recruited all participants, conducted the test visits, and wrote the majority of the manuscript. Ms. Ahn assisted in the collection and the analysis of the data.   The data analyzed in Chapter 4 was collected in the studies outlined in Chapters 2 and 3 of this thesis. I contributed to the conception and design of this retrospective analysis. Mr. Sinden vii  assisted with the data analysis and preparation of the manuscript. Dr. Koehle provided guidance and feedback on the study design, analysis, and preparation of the manuscript. A version of Chapter 4 will be submitted for peer-review.  The research studies outlined in Chapters 2 to 4 were approved by the University of British Columbia’s Clinical Research Ethics Board (H09-01154). viii  Table of Contents Abstract .......................................................................................................................................... ii Lay summary ................................................................................................................................ iv Preface .............................................................................................................................................v Table of contents ........................................................................................................................ viii List of tables....................................................................................................................................x List of figures ................................................................................................................................ xi List of symbols ............................................................................................................................. xii List of abbreviations .................................................................................................................. xiii Acknowledgements  .....................................................................................................................xv Dedication ................................................................................................................................. xviii  Chapter 1: Introduction....…………………………………………………………………........1 1.1 Literature review .....................................................................................................................1 1.2 Purpose, research questions, and hypotheses ........................................................................17  Chapter 2: Effects of inhaled bronchodilators on lung function and cycling performance   in female athletes with and without exercise-induced bronchoconstriction...........................20 2.1 Introduction ...........................................................................................................................20 2.2 Methods.................................................................................................................................21 2.3 Results ...................................................................................................................................25 2.4 Discussion .............................................................................................................................33 2.5 Conclusions and practical implications ................................................................................37  Chapter 3: High-dose inhaled salbutamol does not improve 10-km cycling time trial performance..................................................................................................................................38 3.1 Introduction ...........................................................................................................................38 3.2 Methods.................................................................................................................................40 3.3 Results ...................................................................................................................................43 3.4 Discussion .............................................................................................................................51 3.5 Conclusion ............................................................................................................................56  ix  Chapter 4: Evaluation of the diagnostic criteria of the eucapnic voluntary hyperpnea       test in trained athletes ..................................................................................................................58 4.1 Introduction ...........................................................................................................................58 4.2 Methods.................................................................................................................................61 4.3 Results ...................................................................................................................................67 4.4 Discussion .............................................................................................................................74 4.5 Conclusions ...........................................................................................................................83  Chapter 5: Conclusions ...............................................................................................................84 5.1 Overall conclusions ...............................................................................................................84 5.2 Future directions ...................................................................................................................87 References .....................................................................................................................................95 Appendix .....................................................................................................................................106  x  List of tables Table 1:  Anthropometric, fitness, and spirometric parameters in female athletes with a negative (EVH-) and a positive (EVH+) eucapnic voluntary hyperpnea (EVH) challenge. ...................................................................................................................... 27  Table 2:  Performance parameters measured during the 10-km time trials in female athletes with a positive (EVH+) and a negative (EVH-) eucapnic voluntary hyperpnea (EVH) challenge. ........................................................................................ 31  Table 3:  Anthropometric, fitness, and spirometric parameters in male EVH- and     EVH+ athletes. ............................................................................................................. 45  Table 4:  Correlations between the relative dose of inhaled salbutamol (µg per kg/BM) and spirometric as well as performance parameters. .................................................... 47  Table 5:  Mean and standard deviations for the 2-km time trial interval analysis after inhalation of 1600 µg salbutamol or placebo. .............................................................. 49  Table 6:  Anthropometric, pulmonary, and fitness characteristics of 18 male and           19 female athletes. ........................................................................................................ 68  Table 7:  Respiratory parameters for EVH challenge, maximal graded exercise test,     and time trial. ................................................................................................................ 72  Table 8:  Finishing times and percent differences between the gold, silver, and bronze performances in the road cycling time trials of the past three Olympic Games for male and female athletes. ........................................................................................ 91  xi  List of figures  Figure 1:  Athletic performance after salbutamol and placebo inhalation in athletes   with (EVH+) and without (EVH-) a positive eucapnic voluntary hyperpnea challenge. ...................................................................................................................... 26  Figure 2:  Time effect of salbutamol on cycling and cardiovascular parameters in 2-km intervals. ....................................................................................................................... 29  Figure 3:  Ratings of perceived dyspnea and leg fatigue during 10-km time trial following the inhalation of salbutamol and placebo in female athletes with a positive (EVH+) and negative (EVH-) eucapnic voluntary hyperpnea challenge. ...................................................................................................................... 30  Figure 4:  Rating of perceived dyspnea in male and female athletes following the inhalation of salbutamol and placebo. .......................................................................... 32  Figure 5:  Athletic performance after salbutamol and placebo inhalation in               EVH- athletes (A) and EVH+ athletes (B). .................................................................. 46  Figure 6:  Ratings of perceived dyspnea (A) and perceived leg fatigue (B) displayed as means and standard deviations. .................................................................................... 51  Figure 7:  Number of EVH+ and EVH- tests based on a cutoff of 10% and 15% using the FIA, FIB, FIC, and FID calculation method, respectively. ........................................ 70  Figure 8:  Fall indices calculated using four different methods for every athlete. ....................... 71  Figure 9:  Target ventilation of EVH challenge normalized by ventilation achieved on the graded maximal exercise test.................................................................................. 73   xii  List of symbols ß beta µ micro ≥ greater or equal than ≤ smaller or equal than  xiii  List of abbreviations AAF Adverse analytical finding AHR Airway hyperresponsiveness ATS American Thoracic Society ß2-agonist Beta2-agonist CO2 Carbon dioxide EIA Exercise-induced asthma EIB Exercise-induced bronchoconstriction EIB+ Individual with a positive diagnosis of exercise-induced bronchoconstriction EIB- Individual with a negative diagnosis of exercise-induced bronchoconstriction EELV End-expiratory lung volume EFL Expiratory flow limitation EILV End-inspiratory lung volume EVH+ Individual with a positive EVH challenge EVH- Individual with a negative EVH challenge EVH test Eucapnic voluntary hyperpnea test FEV1 Forced expiratory volume in 1 second FEF50 Forced expiratory flow at 50% of vital capacity FIA Fall Index A FIB Fall Index B FIC Fall Index C FID Fall Index D FVC Forced vital capacity GXT Graded maximal exercise test HR Heart rate IBA Inhaled beta2-agonist ICS Inhaled corticosteroid IOC International Olympic Committee IOS Impulse oscillometry xiv  kg/BM Kilograms per body mass LABA Long-acting beta2-agonist N2 Nitrogen O2 Oxygen SABA Short-acting beta2-agonist rpm Rotations per minute RPEL Rating of perceived exertion for leg fatigue RR Respiratory rate SpO2 Peripheral capillary oxygen saturation TUE Therapeutic use exemption VE Minute ventilation VEEVH-achieved Average minute ventilation achieved during the eucapnic voluntary hyperpnea test VEEVH-target Average target minute ventilation achieved during the final five minutes of the eucapnic voluntary hyperpnea test VEGXT Highest 15 second average for minute ventilation achieved during the final minute of the graded maximal exercise test  VETT Average minute ventilation achieved during the final two kilometers of a    10-km time trial VO2 Oxygen consumption VO2RM Oxygen consumption of the respiratory muscles Vt Tidal volume WADA World Anti-Doping Agency WOB Work of breathing    xv  Acknowledgements Hiking in the backcountry, particularly in British Columbia, is one of my favourite leisure time activities. Looking back on my doctoral training, I like to compare my experiences with that of a hike. There were certainly sections along the way that included some strenuous bushwacking and crossroads that challenged my navigational skills when trying to stick with the path that would take me to the top of the mountain; however, these tough periods were rewarded by many spectacular views along the way. I am very lucky and extremely grateful to have had the guidance, support, and help of my mentors, friends, and family on my “hike” to this PhD. In the following paragraphs, I would like to thank everybody who has accompanied me along the way.   I would like to thank Dr. Michael Koehle, my MSc and PhD supervisor. I consider myself the winner of the lottery to have had the opportunity to study under your supervision. Working in the Environmental Physiology Laboratory provided me with countless opportunities for academic and personal growth. No new study idea seemed impossible and no distance was too far to set up a meeting with you to discuss those ideas. I am grateful for having had the opportunity to work independently to develop my own research and leadership skills, while knowing that you would be there for support should I get stuck. Learning from you has been an absolute privilege, and I consider you a role model not just in academia but also in life in general.  Thank you to my thesis committee members Dr. Jordan Guenette and Dr. Chris Carlsten. I am grateful that you welcomed me in your laboratories and provided me with the opportunity to work in new settings. Thanks to you, I was able to include measurement techniques and data analyses in my studies, which I was previously unfamiliar with. Thank you also for your xvi  guidance and feedback throughout my entire doctoral training on written documents, in committee meetings, or when preparing for conference presentations.  Without the help of the administrative staff in the School of Kinesiology at the University of British Columbia I would have missed several deadlines due to confusion on my behalf on how to fill in important documents, or how to solve challenges with respect to course registrations, reimbursement forms, tuition payments, etc. I would like to sincerely thank everybody in the War Memorial Gym 210 office, particularly Helen Luk and Kathy Manson, for always helping me when the deadlines were approaching way too quickly. Whenever I showed up stressed in the 210 office, I left feeling relieved and with a smile on my face thanks to the help of all of you!  Research studies cannot be completed without the help of lab mates and fellow undergraduate and graduate students. I would like to thank all my colleagues and friends who have helped me make my studies possible by supporting me to recruit participants, assisted in the data collection, helped me with statistics and wrote python codes for me, proofread manuscripts and accepted that my knowledge for periods and hyphens still has potential for improvement. Martin, Luisa, Eric, Meaghan, Maha, Kristin, Assaf, Normand, Sean, Raymond, Rei, Kristina, Joe, and Andrew thank you very much!  Similarly to it being impossible to complete certain hikes by oneself, it would have been impossible for me to complete my PhD without my friends. One of the best “side-effects” of my doctorate is that I won new friends and got to strengthen “old” friendships over the past few years. Katja, Cathy, Erica, and Carolyn, going on this “PhD-hike” together with you meant that I xvii  was in the very best company I could have ever wished for. Thank you for helping me recalibrate my coordinate system when the axes seemed a bit misaligned. We have had so many great laughs together with epic ice cream excursions, and I look forward to all the adventures that we get to share with our PhDs in our pockets now.  Jess and Alex, Babette, Kat, Bieke, Ange and Mike, Ria and Petra, Eric, Kala, SJ, Hildur and Paul, Grant and Tova, Josi, Sandy, Catrin, Eva, Vera, Corinne, Sandy and Philip, Ursina, Nici, Noëlle, my Davis friends, and The Right Shoe Runners, thank you for your friendship and support, for inviting me on all these unforgettable adventures that have allowed me to recharge my batteries when needed, and for the many laughs!  Liebe Mama, Lieber Papa, Lieber Raphael, mit Worten kann ich meine Dankbarkeit Euch gegenüber nicht ausdrücken. Wenn ich an mir gezweifelt habe, ward Ihr es die mich aufgebaut, unterstützt und motiviert haben. Das Wissen über dieses jederzeit vorhandene Auffangnetz, welches Ihr für mich bildet, ist für mich von unschätzbarem Wert! Von Herzen Danke ich Euch fürs immer Dasein, für die vielen Telefongespräche, und die „Unterstütungspäckli“.   xviii  Dedication    For my family Für meine Familie       1  Chapter 1: Introduction  1.1 Literature review  1.1.1 Prevalence, symptoms, and pathogenesis of exercise-induced bronchoconstriction  Exercise-induced bronchoconstriction (EIB) is defined as the transient airway narrowing that occurs as a result of exercise.1 The terms exercise-induced asthma (EIA) and EIB have been used interchangeably, due to a controversy regarding the nomenclature related to bronchoconstriction as a result of exercise.1 Many prefer using the term EIB because it does not imply that affected individuals have underlying chronic asthma. For the remainder of this thesis, the term EIB will be used without regard to whether it occurs in athletes with or without asthma.   Depending on the source cited, approximately 30% - 50% of asthmatics are affected by EIB, but it can also occur in otherwise healthy individuals without a clinical diagnosis of asthma.2 There is a considerable body of literature suggesting that women are at a greater risk of developing asthma compared to men in the general population3,5; however, further research is needed to determine if there is also a sex difference in the prevalence of EIB among athletes. In the general population, women are at a 10.5% greater risk of developing asthma at some point in their lives compared to men.4 It appears that not only sex but also age affect the prevalence of asthma across the life span. Asthma is more common in prepubertal boys compared to girls.4 After puberty the prevalence of asthma and its severity increase significantly in women, with women being more commonly affected by the condition than men by the age of twenty years. After menopause, the gap in asthma prevalence between men and women becomes narrower but does not disappear.3,4,6 Studies investigating the prevalence of EIB in the athletic population find 2  similar trends as for asthma in the general population. Langdeau et al.7 found a higher prevalence of airway hyperresponsiveness (AHR) and exercise-induced respiratory symptoms in adult female athletes compared to male athletes. It is unclear if adolescent male athletes are at a greater risk for EIB compared to female athletes. In their analysis of the EIB prevalence in adolescent athletes, Johansson et al.8 did not identify a sex difference in the prevalence of EIB in 13 - 15 year-olds Swedish athletes. This is in contrast to findings by De Baets et al.9 and Busquets et al.10 Both studies reported a higher prevalence of EIB among girls than boys.9,10  Symptoms of EIB include cough, wheezing, chest tightness and dyspnea.1 McCallister et al.3 report in their review article that men and women experience asthma differently. Despite comparable or better pulmonary function, women tend to report more asthma symptoms and a lower quality of life than men.3 Bronchial reactivity appears to be altered in the follicular phase of the menstrual cycle of premenopausal women.11 An evaluation of 17 well-controlled female asthmatics showed no differences in the concentration of methacholine needed to produce a 20% fall in lung function from baseline (PC20) or lung function assessed by forced expiratory volume in 1 second (FEV1) in the assessments taken one week before and one week after the start of menses; however, symptoms were worse during menstruation. These findings are in contrast to a study by Oguzulgen et al.12, who evaluated lung function at two points in the menstrual cycle (day 7 days) in 11 women with self-reported premenstrual asthma. In this study, PC20 and FEV1 were significantly decreased during the premenstrual period without achieving statistical significance.12 Similarly, Matteis et al.13 reported a greater decrease in FEV1 following the methacholine challenge in the follicular phase when compared to their response in the luteal 3  phase in 10 out of 36 women. Due to a high degree in inter-individual variability this trend this not reach statistical significance.   Compared to the general population, the prevalence of EIB is significantly greater in athletes.14 Approximately 8% of all competing Olympic athletes suffer from EIB, which makes it the most common medical condition among elite athletes.15 Exercise acts as the trigger of EIB; however, whole-body exercise per se is not the direct cause of the airway narrowing, it is the hyperpnea associated with it. Endurance athletes, i.e. those repeatedly sustaining prolonged periods of intense exercise, are predominantly affected by EIB due to the provocative nature of repeated hyperpnea performed in irritant-laden environments.14 During exercise, minute ventilation (VE) increases from  6 L/min to > 200 L/min in elite endurance athletes, which induces a shift from nasal to mouth breathing.2 Nasal breathing is innate to human respiration because it optimally prepares the inhaled air and protects the deeper structures of the respiratory system, such as the airways and lungs. Nasal inspiration increases the temperature of the inspired air to body temperature, increases its humidity, and filters the air by extracting contaminants such as dust, bacteria, and water-soluble gases prior to the inspirate passing into the remaining respiratory system.16-19 The physiological determinants of the relative contributions of nasal and oral breathing during exercise are unclear. It is thought that the ratio of nasal resistance to airflow determines the relative work of breathing between nasal and mouth breathing and thus the switch from nasal to oronasal breathing during exercise.20 The need to heat and humidify the inhaled air is increased when switching from nasal breathing to oronasal breathing, resulting in abnormal heat and water losses within the lower airways.2 A healthy human is thought to consume up to 350 kcal of heat and 400mL of water per day in order to condition the inspired air at moderate 4  conditions.21 Svenson et al.22 showed that the expired water loss was 42% greater by oral expiration when compared to nasal expiration at a minute ventilation of 9.0 L/min in healthy individuals. It is likely that the difference in water loss will be greater at increased ventilations.   Two theories are discussed in the pathogenesis of EIB: the hyperosmolar hypothesis and the thermal (also known as airway cooling) hypothesis.23 According to the hyperosmolar hypothesis, exercise hyperpnea leads to the dehydration of the airway surface liquid through evaporation by humidifying the inspired air.24 Due to the increased osmolarity of the airway mucosal, submucosal, and epithelial layers, bronchoconstriction mediators (leukotrienes, prostaglandins, or histamines) are released from inflammatory cells, leading to the acute narrowing of the airways.2,25 According to the thermal hypothesis, the cooling effect of hyperpnea causes the bronchial circulation to vasoconstrict, which is followed by reactive hyperemia as the exercise ceases and ventilation returns to resting levels. The reactive hyperemia is thought to cause vascular leakage and edema, inducing vascular engorgement in the mucosa that is responsible for the transient narrowing of the airways in EIB.26 To date, there are studies supporting27,28 and disputing29,30 the thermal hypothesis of EIB. Thermal stress does not appear to be a prerequisite for EIB, but it contributes to EIB when exercise is performed in low temperatures because cold inspired air initially increases the surface area of the airways that becomes dehydrated and hyperosmotic, while vascular engorgement could magnify the narrowing effect of smooth muscle contraction in the airways.2   Besides dehydration and thermal stress, mechanical trauma cannot be overlooked in the pathogenesis of EIB. For example, the prevalence of EIB in cross-country skiers (17%) is 5  nearly six-fold greater than in ski jumpers (3%).15 While both groups are repeatedly exposed to cold, dry air, there are marked differences in the total ventilatory demands between the two sports. When air is moved across the surface of the airway, shear stress causes the airway walls to stretch and the epithelial cells to deform due to greater trans-airway pressure.2 Signaling, structural, and mechanical responses that accompany the mechanical deformation of the epithelium31 may impair airway function and cause further trauma to an already inflamed epithelial layer.2 Cough is a common symptom of EIB,32 which is thought to be a contributing factor to airway injury, inflammation, and obstruction due to increases in wall shear stress.33 The wall shear stress during a cough is up to 20 times higher than during regular breathing at rest with relatively low airflows. During exercise, the increased osmolarity of the airway surface liquid likely activates sensory nerves and triggers cough, which may increase AHR and inflammatory levels.25 Interestingly, deep breaths, as they commonly occur in exercise, can also act as potent bronchodilators, and are likely responsible for exercise-induced bronchodilation in non-asthmatics. In healthy individuals, deep breaths are thought to maintain airway patency by detaching smooth muscle crossbridges.2 In asthmatics, this bronchodilatory effect of deep breathing may be impaired.34  1.1.2 Diagnostics The symptoms of EIB are non-specific, meaning that they also occur with other conditions; therefore, diagnoses of EIB should never be based on respiratory symptoms alone, as they are poor predictors of the presence or severity of EIB.14,35 Direct and indirect tests are used to screen for EIB.14 Direct tests are pharmacological tests that induce direct contraction of the airway smooth muscle cells by using agents such as methacholine or histamine. Indirect tests assess the 6  degree of bronchoconstriction induced through dehydration of the airway surface liquid with a subsequent increase in airway surface osmolarity. Indirect tests include the exercise, eucapnic voluntary hyperpnea (EVH), and mannitol challenges. Positive methacholine challenges with negative exercise, EVH, or mannitol tests are commonly encountered in athletes.35 One possible theory for positive direct and negative indirect EIB challenges in the same elite athlete could be injured epithelial cells that enhance the access of methacholine to the muscarinic receptors on the bronchial smooth muscle, and thus enhance bronchoconstriction.36 This (direct) pathway of inducing bronchoconstriction is different from the one employed by indirect challenges. Furthermore, the epithelial airway injury in elite athletes may not be linked to airway inflammation, which could explain the negative mannitol, exercise, or EVH challenges in these same athletes.35,36 As a result, an EIB diagnosis should not be based on a positive methacholine challenge alone, particularly not in individuals without a current clinical history of asthma.14,35   For the purpose of this thesis, the exercise and EVH challenges will be described in further detail. The aim of both challenges is to assess the degree of bronchoconstriction as a response to evaporative water loss due to humidifying large volumes of inspired air in the airways in a short period of time.35 The exercise challenge is designed as a short, intense exercise bout on an ergometer or in the field to generate highest VEs. The EVH challenge includes voluntary hyperpnea with a target VE (VEEVH-target) of 30 x FEV1 at baseline, during which athletes mimic the high VEs they generate during short, intense whole-body exercise. The major factors that determine the severity of EIB in the exercise and EVH challenges are the VEs achieved and sustained, in addition to the water content and temperature of the inspired air.37 The greater the evaporative water loss by bringing large volumes of air to body temperature in a short time, the 7  more likely the airways are to constrict. To avoid false-negative tests, individuals participating in an exercise or EVH challenge are asked to avoid exercise and asthma medication intake up to 12 hours prior to the test because of a refractory period that could inhibit the production or release of bronchoconstriction mediators.35 A diagnosis of EIB (EIB+) is typically based on a decrease in FEV1 ≥ 10% during or after the exercise or EVH challenge.35 The airway response is expressed as the percent change between the highest FEV1 value at baseline and the lowest value after the diagnostic challenge. This percent difference in FEV1 is called the fall index (FI). The severity of EIB is graded as mild if FI ≥ 10% but < 25%, moderate if FI ≥ 25% but < 50%, and severe if FI ≥ 50%.1  Exercise challenge Exercise challenges consist of rapidly ramped protocols, without prior warm-up, lasting six to eight minutes. To induce the greatest possible evaporative water loss of the airway surface liquid, participants should breathe dry air via the mouth and exercise at an intensity ≥ 85 % of maximal heart rate or 21 x baseline FEV1 four minutes into the test, preferably within the first two minutes.14,35 Since exercise loads determine the intensity of the bronchoconstriction stimulus, standardized protocols for exercise challenges are needed.38 A difference of 10% in exercise intensity, set to 85% and 95% of the predicted maximal heart rate (calculated as 220 - age), led to a significant difference in the number of athletes with a positive exercise challenge.38 In the Therapeutic Use Exemption (TUE) Physician Guidelines on asthma provided by the World Anti-Doping Agency (WADA) in 2015,39 a FI of 10% is recommended for a positive exercise challenge; however, detailed instructions on how to standardize exercise challenge protocols for various sport disciplines are lacking. Furthermore, despite sport governing bodies requiring 8  specific cutoff values to diagnose EIB, there is no single absolute cutoff for a decrease in FEV1 that clearly and indisputably distinguishes between an EIB+ and EIB- diagnosis.14,40 Cutoffs of decreases in FEV1 ranging between 6% – 15% for laboratory-based tests, and 15% for field-based tests have been recommended.14 When using a standardized protocol under well-controlled conditions, the exercise challenge has been shown to be reproducible in individuals with mild symptoms of asthma.41 Besides the standardization of the exercise protocol and the cutoff value distinguishing between a positive and negative challenge, previous medication intake, the duration between the last exercise bout and the exercise challenge, and seasonal factors such as allergens, pollutants, air temperature, and humidity have the potential to influence the result of an exercise challenge.35 In an attempt to minimize the effect of those factors that are difficult to control and known to influence the bronchoconstriction potential, the EVH challenge has become a preferred surrogate of the exercise challenge in the diagnosis of EIB.   EVH challenge The EVH challenge was introduced as a diagnostic test for EIB by members of the US Army42-44 and is considered the best available laboratory test to diagnose EIB by the International Olympic Committee.45 The protocol of the EVH challenge consists of the hyperventilation of compressed dry mixed gas (5% carbon dioxide (CO2), 21% oxygen (O2) balanced with nitrogen (N2)) at room temperature for six minutes.46 The CO2 is included in the inspirate to maintain the arterial CO2 levels within the range documented during exercise. Spirometry is performed in duplicate at 3, 5, 10, 15, and 20 minutes after hyperpnea. An FI ≥ 10% is considered indicative of EIB+ with a specificity of 90% and a sensitivity of 63%.1,14,35,42 Similarly to the exercise challenge, the FI of the EVH challenge is calculated as the percent decrease between the highest FEV1 value assessed 9  at baseline and the lowest of the five post-challenge values, always taking the higher of the measures in duplicate into account for every time point. Some of the advantages of the EVH challenge over the exercise test in the diagnosis of EIB in athletes are: (1) The VEEVH-target is calculated as a product of FEV1 and independent of the locomotive muscle power of athletes;   (2) the protocol of the EVH challenge can easily be adapted with respect to duration of the eucapnic hyperpnea and the temperature of the inspired air to simulate the conditions under which exercise is performed.35  Since its introduction, modifications to the calculation methods of the FI, specifically the determination of the lowest FEV1 measure post-hyperpnea, have been suggested to improve the diagnostic accuracy of the EVH challenge. These suggestions include:   (1) Normalization of FI by achieved VEs during EVH challenge Because the intensity of the bronchoconstriction stimulus generated by the EVH challenge is determined by the achieved VE (VEEVH-achieved), Argyros et al.44 and Hurwitz et al.42 suggested that the FI should be corrected by the VEEVH-achieved during the 6-minute hyperpnea period. As previously mentioned, the VEEVH-target is typically calculated as 30 x FEV1 at baseline;46 however, not all test takers are able to reach these VEs due to unachievably high targets, fatigue, hesitation from the anticipation of bronchoconstriction, or lack of motivation.   (2) Basing an EVH+ challenge on an FI ≥ 10% at two consecutive time points as opposed to one time point only. 10  The purpose of this amendment to the original EVH challenge protocol is to determine whether a decrease in FEV1 is sustained and representative of the transient narrowing of the airways, rather than an artifact representing poor technique, sub-optimal effort, or fatigue.14,35    (3) Introduction of a diagnostic cutoff value of FI ≥ 15% as opposed to an FI ≥ 10%  Recently, the use of 15% as a cutoff was suggested, based on the typical definition of an abnormal response as the mean and two standard deviations of the decline in FEV1 in response to EVH in healthy individuals.47 Price et al.47 explain that 44/224 (i.e. 20%) competitive asymptomatic athletes responded to the EVH challenge with a fall in FEV1 close to or beyond 10%, representing a normal, healthy response.   1.1.3 Treatment Pharmacological and non-pharmacological treatment strategies exist for EIB. Short-acting ß2-agonists (SABAs) and long-acting ß2-agonists (LABAs) are commonly used as preventive or acute treatments for EIB.1,48 If symptoms are frequent and the use of SABAs and LABAs insufficient on an as-needed basis, daily inhaled corticosteroids (ICS) are added. Other commonly used medications are leukotriene antagonists and mast cell stabilizers, either as a single medication or in combination with others. Adding a warm-up to a training session to induce a refractory period,49 and efforts to pre-warm and humidify inspired air during exercise by breathing through a face mask or a scarf, are commonly used non-pharmacological methods to reduce the symptoms of EIB.1  11  The literature review in this thesis will focus on the role of inhaled β2-agonists (IBAs) in the treatment of EIB symptoms and their effects on athletic performance.  Inhaled β2-agonists and its use in sport More changes have been made to the status of inhaled salbutamol on WADA’s Prohibited List than to any other substance in the past 45 years.50 β2-agonists, such as salbutamol, represent a class of medications that are commonly used in symptomatic and non-symptomatic athletes with mild EIB.1,51 These agents act as powerful bronchodilators by reducing airflow obstruction; however, they also have the potential to affect parameters known to limit athletic performance, such as heart rate (HR), blood flow through the coronary and peripheral arteries, and substrate availability.52 By mimicking epinephrine, β2-agonists act on the adrenergic β2-receptors, which are located primarily in the lungs but also in the heart and the skeletal muscles. In the lung, β2-agonists widen the airways by inducing smooth muscle relaxation in the cells surrounding the airways.52 In the heart and the skeletal muscles, β2-agonists vasodilate the arteries, increasing blood flow. Furthermore, β2-agonists increase heart rate and contractility and improve substrate availability by stimulating glycogenolysis and glycolysis.52 Additionally, increased levels of cyclic adenosine monophosphate to β2-agonist use have been linked to an enhanced lung fluid clearance via augmented Na+ transport across alveolar epithelial cells.53,54 Lastly, through their stabilizing effect on mast cells and inhibition of mediator release from eosinophils, macrophages T-lymphocytes, and neutrophils, β2-agonists are also thought to have anti-inflammatory effects.55,56  The most common form of inhaled β2-agonist (IBA) intake among athletes is through a metered dose aerosol, with or without a spacer.50 Due to the anabolic effect associated with athletic 12  performance improvements when taken systemically, all β2-agonists are on the WADA list of prohibited substances, except inhaled salbutamol (maximum: 1600 μg, without exceeding 800 μg every 12 hours), inhaled formoterol (maximum: 54 μg over 24 hours), and salmeterol (maximum: 200 μg over 24 hours).39 If excessive amounts of salbutamol (> 1000 ng/mL) or formoterol (> 40 ng/mL) are detected in urine samples, the intake is presumed not to be of therapeutic nature and is considered an Adverse Analytical Finding (AAF) by WADA, unless the athlete can prove that the abnormally-high urine levels were the result of an inhaled therapeutic dose.39 As of January 1st, 2010, the requirement of applying for a Therapeutic Use Exemption (TUE) prior to IBA use, and as of January 1st, 2011, the requirement of sending a Declaration of Use form to anti-doping organizations have been lifted.39 Currently, athletes are instructed to write all medications (including IBAs) and substances taken in the last seven days on the doping control form at the time of testing.   Interestingly, athletes on IBAs have won a disproportionately greater percentage of individual Olympic medals compared to athletes who are not taking IBAs.15 In the past 25 years, there has been an increase in applications for TUEs and notifications of use of IBAs from Olympic-level athletes.15 For example, at the Los Angeles Olympic Games (1984) the prevalence of IBA use was 1.7% and has gradually risen since: Atlanta (1996) 3.7%, Nagano (1998) 5.6%, Sydney (2000) 5.7%, Torino (2002) 7.7%, Beijing (2008) 6.1%.50,57 In these past Olympics Games, these IBA users have won a disproportionate number of medals. For example, in Vancouver, 7.1% of the athletes used IBAs and won 11.8% of the medals.15 These results beg the question whether IBAs have an ergogenic effect.   13  Anti-doping regulations and research on the ergogenic potential of IBAs Pluim et al.58 and Kindermann59 concluded in their meta-analyses that IBAs do not have a significant effect on endurance performance in athletes without EIB. In agreement with this conclusion, our group demonstrated that despite a significant improvement in FEV1 following 400 μg of inhaled salbutamol, cycling performance was not improved in trained male cyclists, regardless of bronchial hyperresponsiveness.60 The majority of studies investigating the effects of IBAs on athletic performance have been conducted in men.60-62 It is unclear if women’s respiratory responses to IBAs, and the subsequent effects on parameters relevant for athletic performance, are comparable to those in men. Furthermore, limited studies have looked at the effects of the maximally allowed daily doses of IBAs on athletic performance, particularly when taking relative IBA dose into account by normalizing the IBA dose by body mass (BM).   Sex differences in the respiratory response to IBAs and athletic performance  A number of anatomical and functional sex differences are responsible for differing respiratory responses to exercise between men and women.63-65 Dysanapsis refers to the unequal growth and physiological variation between airway size and lung volume.66-69 When matched for height, women have smaller lung volumes and smaller diffusion surfaces for pulmonary gas exchange compared to men.64,70 When matched for lung size, women have smaller airway luminal areas and decreased conducting airway diameters compared to men.69 As a result of these sex-based anatomical differences, women have lower maximal expiratory flows for a given lung volume, leading to a reduced ventilatory capacity, which makes women more susceptible to mechanical ventilatory constraints during exercise.64,71,72 14  To sustain elevated power outputs during high-intensity exercise, elite athletes require high VEs generated by increased airflow in maximally dilated airways. Many athletes demonstrate an upper limit in their ability to generate expiratory flow, called expiratory flow limitation (EFL).73 Trained women have been considered more susceptible to EFL than trained men, due to the anatomical and functional sex differences in the respiratory system outlined above;65,71,72 however, this remains a controversial topic. In a study on recreational, less fit individuals, men and women were at equal risk to develop EFL.74 Those with a low dysanaptic ratio (i.e. those with a low ratio between airway and lung size) were most likely to develop EFL during exercise, independent of biological sex.74 A recent study showed an effect of fitness on the prevalence of EFL.75 Differentiating between high and low fitness levels, fit female athletes experienced EFL more often compared to fit men; whereas among less-fit athletes, a sex difference did not seem to prevail.75  In an effort to use higher airflows that are available at larger lung volumes when experiencing EFL, end-expiratory and end-inspiratory lung volumes (EELV and EILV, respectively), as well as pleural pressures increase, resulting in substantially more energy required to maintain high VEs.65 The energy necessary to meet the metabolic demands of the respiratory system is called work of breathing (WOB). Total WOB is calculated as the sum of the following four components: inspiratory muscle work to overcome the elasticity of the lung and to overcome airflow resistance during inspiration, the expiratory muscle work to overcome the elastic outward recoil of the chest wall, and the work to overcome airflow resistance during expiration.76 Guenette et al.71 showed a higher total WOB for a given absolute VE > 60 L/min in women 15  compared to men. In a subsequent study, the higher WOB was attributed to a higher resistive versus elastic WOB, which is related to their narrower airways.76  Anatomical and functional sex differences leading to variations in the pulmonary response to exercise may allow women to have a greater potential to improve athletic performance when using bronchodilators such as IBAs. According to Poiseuille’s law, airway diameter affects airflow by the fourth power; therefore, an IBA-induced bronchodilatory effect in women could lead to greater relative improvements in FEV1 and possibly athletic performance by reducing the resistive WOB and delaying or cancelling the onset of EFL compared to men. Harms et al.77 reduced the inspiratory muscle work during maximal cycling exercise with a proportional assist ventilator. He showed that the proportion of total VO2 available to the legs was increased from 81% in the control condition to 89% in the unloading condition. Building on their conclusion that increases in WOB during maximal exercise compromises locomotor muscle perfusion and VO2 begs the question if a potential ergogenic mechanism of IBAs can be explained with a decrease in WOB via bronchodilation, thus facilitating perfusion and increasing the proportion of total VO2 available in the locomotor muscles.  Maximal daily doses of IBAs on athletic performance Studies investigating the ergogenic potential of therapeutic and supratherapeutic doses of inhaled salbutamol ranging from 200 µg to 800 μg did not show an effect on athletic performance.60-62 Few studies have investigated the effects of the maximally allowed daily dose of IBAs on athletic performance. Dickinson et al.78 reported an increase in VE of 10 L/min at every kilometer over a 5-km distance following the inhalation of 1600 μg of salbutamol in runners; however, this increase in VE did not reach statistical significance, nor did it affect running 16  performance. In a second study by Dickinson et al.79 investigating the effects of a six-week loading period using 1600 μg of salbutamol per day, there were no differences observed in peak oxygen consumption, 3-km running performance, or one-repetition maximum for bench and leg press.79 The extent to which the results from the two studies by Dickinson et al.78,79 can be extrapolated to elite athletes is debatable due to a small sample size (n = 7),78 a heterogeneous athletic background of the study participants (runners soccer players, cyclists, boxers, etc.),79 and relatively low cardiorespiratory fitness levels.78,79 In contrast to the findings by Dickinson et al.78,79 are the results of a study examining the combined maximal dose of multiple IBAs in swimmers with and without AHR.80 Swim ergometer performance, but not exhaustive swim performance, was significantly improved after the inhalation of 1600 μg salbutamol, 200 μg salmeterol, and 36 μg formoterol, regardless of AHR.80 Thus, even though there does not seem to be an ergogenic effect for salbutamol at low or moderate doses,60-62 there is a potential for the maximally allowed daily dose (i.e. 1600 μg of inhaled salbutamol) to be ergogenic in trained athletes. Furthermore, it is unclear if there is a relationship between the relative IBA dose (i.e. dose per kilogram of body mass, kg/BM) and ergogenic potential. The maximally allowed daily dose of IBAs are not normalized by body mass in the WADA guidelines;39 therefore, lighter athletes may benefit from a considerably greater IBA dose per kg/BM compared to heavier athletes.      17  1.2 Purpose, research questions, and hypotheses Chapter 2:  Purpose:  (1) To investigate the ergogenic effect of IBAs in trained female athletes.    (2) To investigate sex differences in the response to IBAs on lung function and athletic performance in trained male and female athletes.   Research Questions: (1) Do IBAs induce an ergogenic effect in trained female athletes?    (2) Are there differences in the response to IBAs in lung function and athletic performance between trained male and female athletes?   Hypotheses:  (1) Trained female athletes will experience an ergogenic effect following IBA use which will be assessed by a significant increase in mean power output.   (2) Lung function will improve following IBA use in trained male and female athletes; however, only in female athletes will an increase in lung function translate to an improved mean power output.  Chapter 3: Purpose:  To assess the ergogenic potential of the maximally allowed daily dose of one IBA, salbutamol, in trained male athletes.  Research Questions: (1) Does the inhalation of the maximally allowed daily dose of salbutamol induce an ergogenic effect in trained male athletes? 18  (2) Is the ergogenic effect of IBAs dependent on the relative dose, i.e. the dose per kg/BM?  Hypotheses:  (1) There will be an ergogenic effect on mean power output in trained male athletes following the inhalation of the maximally allowed daily dose of salbutamol.   (2) The increases in athletic performance following the inhalation of the maximally allowed daily dose of salbutamol will be more pronounced in athletes with a lower mass compared to athletes with a greater mass, due to an increased dose per kg/BM.   Chapter 4: Purpose:  (1) To assess four previously published calculation methods of the FI in the EVH challenge in trained athletes.  (2) To compare the bronchoconstriction potential generated by the EVH challenge to the bronchoconstriction potential of a short, intense exercise bout and a graded maximal exercise test by comparing VEEVH-target and VEEVH-achieved to the VEs of a time trial and a graded maximal exercise test.  Research Questions: (1) Do different strategies of assessing the lowest FEV1 among the spirometric assessments post-hyperpnea affect the FI of the EVH challenge? 19   (2) How do VEEVH-target and VEEVH-achieved compare to the VEs of a time trial and a graded maximal exercise test?  Hypotheses: (1) Different methods of calculating the FI will alter the number of positive and negative EVH tests.  (2) The VEEVH-target will be greater than the VE of a time trial. 20  Chapter 2: Effects of inhaled bronchodilators on lung function and cycling performance in female athletes with and without exercise-induced bronchoconstriction  2.1 Introduction As previously described, EIB affects many endurance athletes.15 For example, 17% of cyclists competing at the 2004 and 2008 Olympics treated EIB-symptoms (e.g. coughing, chest tightness, and dyspnea) with IBAs.15 Inhaled β2-agonists act on the adrenergic β2-receptors, which are located primarily in the lungs but also in the heart and the skeletal muscles. In the lung, IBAs act as bronchodilators by inducing smooth muscle relaxation in the cells surrounding the airways.52 In the heart and skeletal muscles, IBAs vasodilate the arteries; therefore, increasing blood flow.  Interestingly, athletes treating their EIB-symptoms with IBAs have won a disproportionately greater percentage of individual Olympic medals as compared to athletes without EIB.15 Pluim et al.58 and Kindermann59 concluded in their meta-analyses that IBAs do not have a significant effect on endurance performance in athletes without EIB. In agreement with this conclusion, our research group recently demonstrated that despite a significant improvement in FEV1 following IBA use, cycling performance in trained male cyclists was not affected, regardless of bronchial hyperresponsiveness.60   Despite the fact that sex-based anatomical differences in the airways and lungs cause differing respiratory responses to exercise in men and women,70,81 female athletes have been generally overlooked in the research on the effects of IBAs on athletic performance. Dysanapsis refers to the altered relationship between airway size and lung volume.66-68 When matched for lung size, 21  women have smaller airway luminal areas and decreased conducting airway diameters compared to men.69 Furthermore, women have smaller lung volumes, smaller maximal expiratory flow rates, and decreased diffusion surfaces relative to men.65,70 During heavy exercise, endurance-trained women were more likely to develop EFL compared to endurance-trained men (90% vs. 43%, respectively).71 In addition, the WOB was significantly greater during high-intensity cycling in female athletes compared to male athletes.71 Since airway diameter affects airflow by the fourth power, an IBA-induced bronchodilatory effect in women could lead to greater relative improvements in FEV1 and athletic performance compared to men.   The primary aim of this study was to investigate the ergogenic potential of IBAs on lung function and athletic performance in trained female endurance athletes with and without EIB. To investigate sex differences in the response to IBAs on lung function and athletic performance, we adhered to the design of one of our previous studies on the effects of IBAs on cycling performance in male athletes.60 We hypothesized that female athletes would demonstrate a significant bronchodilatory response to inhaled salbutamol, which would translate to improved lung function and time trial performance.  2.2 Methods 2.2.1 Preliminary remark As detailed in the preface to this document, we screened and tested female cyclists and triathletes to investigate the ergogenic effects of IBAs in this understudied population. The study design and data collection methods are outlined in the subsequent sections of this Chapter. To assess sex differences in the response to IBAs on lung function and athletic performance, we compared the 22  data collected for this study in female athletes to a previously collected data set from male athletes.60 The study design, data collection procedures, and equipment were identical for both studies. In section 2.2.5, we describe the statistical methods used to analyze the two data sets for sex differences. The results of the sex comparisons are presented in section 2.3.4 and discussed in section 2.4. Participant characteristics and lung function and performance responses to IBA use in male athletes can be found in the manuscript published in the British Journal of Sports Medicine.60  2.2.2 Study participants The 27 screened female cyclists and triathletes of this study were between 19 and 39 years old. Athletes with a maximal oxygen consumption (VO2max) ≥ 50mL/kg/min were included. All athletes were required to have a racing history of at least one year and were participating in or training for races during the data collection. Participants were free of cardiopulmonary disease (excluding controlled asthma) and were not pregnant. Women were tested at random points throughout their menstrual cycle. The use of oral contraceptives was not an exclusion criterion, as hormones relevant to ventilation that can be influenced by estrogen and progesterone are variable between and within women throughout the menstrual cycle, with no effect on submaximal exercise ventilation.82,83  The University of British Columbia Clinical Research Ethics Board provided ethical approval (H09-01154) conforming to the Declaration of Helsinki and written informed consent was obtained from all subjects prior to data collection.   23  2.2.3 Screening visit On the screening visit, bronchial hyperresponsiveness was assessed using the EVH test.46 Lung function was measured with spirometry84 (TrueOne 2400; ParvoMedics, Sandy, UT, USA), and the highest FEV1 from three maneuvers was used as baseline. Athletes then hyperventilated dry gas (5% CO2) for six minutes and repeated spirometry at 3, 5, 10, 15, and 20 minutes post-hyperventilation. A decrease in FEV1 ≥ 10% relative to baseline was classified as EVH+.46 Maximal oxygen consumption was determined during a graded maximal exercise test (GXT) on a cycle ergometer (Velotron Dynafit Pro, RacerMate Inc., Seattle, WA, USA). Participants performed a 10-minute, self-selected warm-up. The GXT began at 0 W, and work rate increased by 0.5 W/second until cycling cadence dropped below 60 rpm.   2.2.4 Time trial visit In this repeated-measures study design, each athlete performed two simulated 10-km time trials, separated by a minimum of three and maximum of 14 days. Athletes were asked to withhold from β2-agonists for at least 12 hours prior to arrival but were allowed to continue corticosteroid treatments. Additionally, athletes did not exercise the day of testing prior to their visit to the laboratory to prevent exercise-induced bronchodilation and did refrain from caffeine consumption. All tests were performed in the same laboratory, in the same season of the year.   On the time trial visits, the effect of each treatment on lung function was assessed by having athletes perform three FEV1 maneuvers prior to, and 30 minutes after, the inhalation of salbutamol or placebo (APOTEX Inc., Toronto, ON, Canada). The treatments were delivered in a randomly assigned, double-blind manner. Prior to the time trials, all athletes performed a self-24  selected, 20-minute warm-up. To assess metabolic parameters during time trials, athletes wore a facemask (Hans Rudolph; Shawnee, KS, USA) connected to a metabolic cart (TrueOne 2400, ParvoMedics, Sandy, UT, USA). A time trial course (3D Cycling, RacerMate Inc., Seattle, WA, USA) was shown on a screen with distance, cadence, and gearing information displayed throughout each time trial. Every 2 km, athletes rated dyspnea and perceived exertion for leg discomfort (RPEL)  on a 0 - 10 Borg-scale.85 The main outcome variable was mean power output over the duration of the 10-km time trial. Secondary outcome variables were cycling economy (ratio of mean power output and mean VO2) maintained over the 10-km time trial in W/L/min, respiratory exchange ratio (RER), HR, VO2, VE, tidal volume (Vt), respiratory rate (RR), dyspnea, and RPEL. To assess for a possible time effect of salbutamol on the outcome variables, these parameters were averaged for each 2-km interval.  2.2.5 Statistical analysis All data are presented as means and standard deviations (M (SD)). Anthropometric, baseline fitness, and spirometric parameters were compared between athletes with and without EIB using the independent samples t-test with EVH status as the grouping variable. The effects of drug treatment and EVH status on athletic performance measures were determined with repeated-measures analysis of variance (ANCOVA) tests with EVH status serving as the covariate. The Shapiro-Wilks W test for normality and Levene test for equal variances were used to confirm that the assumptions of ANCOVA were met. Post-hoc analyses were performed using Tukey’s HSD test if a main effect was detected. We used Pearson product-moment coefficient to assess if fitness levels (i.e.VO2) or the severity of bronchoconstriction (decrease in FEV1 on the EVH test) 25  affected the percent change in mean power output between the placebo and salbutamol time trials. To compare the effects of salbutamol on lung function and time trial performances between women and men, the percent change of all parameters between the salbutamol and the placebo time trials were calculated and then analyzed using one-way ANCOVA tests, where sex served as the covariate. Additionally, we assessed if the ratings of perceived dyspnea provided for every 2-km bout of the two time trials differed between men and women using a repeated-measures ANCOVA with sex as the covariate. Statistical analyses were completed using SPSS (IBM, Version 22.0, Armonk, NY, USA) and statistical significance was accepted when p < 0.05.   2.3 Results 2.3.1 Participant characteristics Of the 27 female athletes screened, 21 met all inclusion criteria and were included in the study. Based on a positive EVH test, six athletes were classified as EVH+. The 15 EVH- athletes (30 (5) years) were significantly older compared to the EVH+ athletes (24 (4) years, p = 0.030). There were no differences between EVH+ and EVH- athletes in height, mass, fitness, or baseline spirometry. These data are comprehensively reported in Table 1.  By definition, EVH+ athletes showed a significantly greater drop in FEV1 (16.3 (3.3)%) after the EVH test compared to EVH- athletes (6.6 (1.8)%, p < 0.001). The bronchial hyperresponsiveness of all six EVH+ athletes was classified as mild (percent decrease in FEV1 ≥ 10% but < 25%). Only two EVH+ athletes had previously been diagnosed with EIB: these athletes were treating their symptoms with daily ICS and IBAs on an as-needed basis. Two EVH+ athletes had no 26  previous diagnosis of EIB but reported (prior to the EVH test) that they experienced difficulty breathing during high-intensity workouts. Two other EVH+ athletes did not report any respiratory symptoms when training and were not aware of their bronchial hyperresponsiveness. One EVH- athlete had childhood asthma but did not respond to the EVH test (percent decrease in FEV1 = 6.6 %).  2.3.2 The effect of IBAs on lung function Lung function, assessed by FEV1, significantly improved 30 minutes after the inhalation of salbutamol (4.6 (4.7)%) compared to 30 minutes post-placebo inhalation (-0.1 (2.8)%,                    p = 0.002). The bronchodilatory response to inhaled salbutamol did not differ statistically between EVH+ (6.1 (7.6)%) and EVH- athletes (4.0 (3.1)%).   Figure 1: Athletic performance after salbutamol and placebo inhalation in athletes with (EVH+) and without (EVH-) a positive eucapnic voluntary hyperpnea challenge.  The dashed line represents the mean. 27  Table 1: Anthropometric, fitness and spirometric parameters in female athletes with a negative (EVH-) and a positive (EVH+) eucapnic voluntary hyperpnea (EVH) challenge. Parameter Total (n = 21) EVH– (n = 15) EVH+ (n = 6) Mean (SD) Range Mean (SD) Range Mean (SD) Range Age (years) 28 (6) 19 – 39 30 (5) 19 – 39 24 (4)a 19 – 28 Height (cm) 167 (6) 157 – 175 167 (6) 157 – 175 166 (7) 157 – 175 Mass (kg) 61 (5) 54 – 68 61 (4) 54 – 68 61 (5) 55 – 68 Cycling experience (years) 5 (5) 1 – 20 6 (5) 1 – 20 3 (3) 1 – 7 Cycling volume (km/week) 198 (61) 100 – 300 209 (52) 100 – 300 174 (78)  75 – 300 FVC (L) 4.8 (0.6) 3.7 – 6.2 4.9 (0.7) 3.7 – 6.2 4.6 (0.2) 4.2 – 4.8 FVC predicted (%) 120 (14) 102 – 155 122 (14) 105 – 155 114 (10) 102 – 129 FEV1 (L) 3.9 (0.6) 2.8 – 5.5 3.9 (0.7) 2.8 – 5.5 3.7 (0.4) 3.0 – 4.1 FEV1 predicted (%) 113 (14) 91 – 159 116 (16) 91 – 159 107 (12) 94 – 122 FEV1/FVC (%) 81 (6) 70 – 91 81 (6) 70 – 90 81 (7) 71 – 91 FEV1/FVC predicted (%) 94 (7) 80 – 105 94 (7) 80 – 105 93 (7) 81 – 103 Δ Max FEV1 (%) 9.4 (5) 3 – 22 6.6 (1.8) 3 – 9 16.3 (3.3)b 13 – 22 VO2max (mL/kg/min) 54.2 (3.4) 50.0 – 63.1 54.2 (3.0) 50.0 – 62.7 54.2 (4.7) 50.5 – 63.1 Maximal power (W) 324 (13) 297 – 346 325 (12) 297 – 346 323 (15) 300 – 337 Maximal power (W/kg) 5.3 (0.4) 4.6 – 6.1 5.4 (0.4) 4.6 – 6.1 5.3 (0.3) 4.9 – 5.6 FEV1, forced expiratory volume in one second; FVC, forced vital capacity; SD, standard deviation; VO2max, maximal oxygen consumption, Δ Max FEV1 decrease in FEV1 in response to a eucapnic voluntary hyperpnea test. a EVH+ athletes were significantly younger than EVH- athletes (p = 0.030). b The decrease in FEV1 in response to the EVH challenge was significantly greater in EVH+ compared to EVH- athletes (p < 0.001). 28  2.3.3 The effect of IBAs on athletic performance The inhalation of 400 µg of salbutamol led to a significantly reduced 10-km time trial performance in trained female athletes (p = 0.047), regardless of athletes’ EVH status (Figure 1). Mean VO2 during the time trial was significantly increased after the inhalation of salbutamol compared to placebo (p = 0.049). Consequently, cycling economy of the entire 10-km time trial was significantly decreased following salbutamol compared to placebo (p < 0.001). The inhalation of salbutamol significantly reduced RER compared to placebo in the 0- to 2-km interval (p = 0.043). In all other intervals, RER remained decreased after the inhalation of salbutamol compared to placebo, but the differences did not reach statistical significance. The time effect of salbutamol on all primary and secondary parameters is shown in Figure 2.   Five (two EVH+ and three EVH-) athletes increased their performances by more than 1% relative to placebo (range: 2% – 9.0%) and 10 (two EVH+ and eight EVH-) athletes decreased their performances by more than 1% relative to placebo (range: -1.2% – -19.6 %). Neither the severity of bronchial hyperresponsiveness assessed by the percent decrease in FEV1 in the EVH test (r = -0.092, p = 0.811), nor fitness levels assessed by VO2max on the assessment day              (r = 0.423, p = 0.103), correlated with the change in mean power output after the two time trials. The percent change in mean power output positively correlated with the percent change in VE between the two time trials (r = 0.474, p = 0.031). None of the additional cardiovascular parameters were altered by the salbutamol treatment (Table 2). The ratings of perceived dyspnea and leg fatigue did not differ across conditions for any time point (Figure 3).    29   Figure 2: Time effect of salbutamol on cycling and cardiovascular parameters in 2-km intervals. * indicates that a significant main effect of salbutamol. Post-hoc testing using Tukey’s HSD identified the 2-km intervals at which statistically significant differences between the salbutamol and placebo condition existed between the two means.   30   Figure 3: Ratings of perceived dyspnea and leg fatigue during 10-km time trial following the inhalation of salbutamol and placebo in female athletes with a positive (EVH+) and negative (EVH-) eucapnic voluntary hyperpnea challenge.   2.3.4 Comparison of IBA-induced effects on lung function and athletic performance between female and male athletes When we compared our data from the current study with those from the identical protocol in 49 male athletes,60 the effects of salbutamol on FEV1 30 minutes after inhalation were similar with 4.6 (4.7)% in women and 6.1 (4.7)% in men, p = 0.861.  31  Table 2: Performance parameters measured during the 10-km time trials in female athletes with a positive (EVH+) and a negative (EVH-) eucapnic voluntary hyperpnea (EVH) challenge. Parameter Total (N = 21) EVH- (N = 15) EVH+ (N = 6) Mean (SD) Range Mean (SD) Range Mean (SD) Range Power (W) Salbutamol Placebo  204 (21)a 208 (17)  167 – 238 173 – 240  207 (20) 210 (15)  167 – 238 183 – 240  197 (25) 201 (22)  168 – 227 173 – 228 VO2 (mL/kg/min) Salbutamol Placebo  46.9 (5.9)b 44.8 (4.0)  36.0 – 58.9 35.7 – 51.3  47.8 (6.0) 45.9 (3.6)  36.0 – 58.9 39.3 – 51.3  44.8 (5.5) 42.0 (3.7)  40.4 – 55.3 35.7 – 46.5 Economy (W/L/min) Salbutamol Placebo  72.8 (6.8)c 76.4 (4.3)  60.4 – 82.3 69.8 – 83.7  72.9 (6.8) 75.5 (4.1)  60.4 – 82.3 69.8 – 83.7  72.5 (7.4) 78.6 (4.6)  69.8 – 83.3 73.4 – 83.7 RER  Salbutamol Placebo 0.99 (0.07) 1.01 (0.07) 0.81 – 1.07 0.86 – 1.10 0.99 (0.08) 1.01 (0.07) 0.81 – 1.07 0.86 – 1.10 0.97 (0.08) 1.02 (0.08) 0.87 – 1.06 0.90 – 1.10 Heart rate (b/min) Salbutamol Placebo  169 (12) 166 (11)  142 – 181 142 – 182  165 (12) 164 (10)  142 – 181 145 – 180  178 (4) 170 (15)  170 – 181 142 – 182 Ventilation (L/min) Salbutamol Placebo  96 (16) 94 (16)  70 – 122 64 – 128  98 (17) 96 (14)  70 – 122 72 – 128  91 (14) 89 (20)  74 – 111 64 – 120 Respiratory rate (b/min) Salbutamol Placebo  46 (9) 46 (10)  34 – 63 27 – 61  46 (10) 46 (11)  34 – 63 27 – 61  45 (4) 46 (7)  40 – 51 38 – 56 Tidal volume (L) Salbutamol Placebo  2.1 (0.3) 2.1 (0.4)  1.7 – 3.0 1.7 – 3.2  2.2 (0.3) 2.2 (0.4)  1.7 – 3.0 1.7 – 3.2  2.0 (0.1) 1.9 (0.2)  1.9 – 2.2 1.7 – 2.2 EVH: eucapnic voluntary hyperpnea, RER: respiratory exchange ratio; VO2: oxygen consumption. a Mean power output was significantly decreased in the salbutamol time trial compared to the placebo time trial, p = 0.047.  b Mean VO2 was significantly increased in the salbutamol time trial compared to the placebo time trial, p = 0.049. c Cycling economy was significantly decreased in the salbutamol time trial compared to the placebo time trial, p = 0.010. 32  There were no differences in the percent change in power output maintained during the two time trials between men (-0.4% (3.4)) and women (-2.1% (6.1), p = 0.172); however, there was a significant interaction between VO2 and sex (p = 0.033) as well as in cycling economy and sex  (p = 0.002). In men, mean VO2 was similar in the salbutamol (55.9 (6.4) mL/kg/min) and placebo (56.1 (6.5) mL/kg/min, p = 0.712) time trials, but in women VO2 was significantly increased after salbutamol compared to placebo (see Table 2). In addition, cycling economy was similar in the salbutamol (72.2 (5.5) W/L/min) and placebo (72.3 (5.5) W/L/min, p = 0.731) time trials in men, but in women cycling economy was significantly decreased after the inhalation of salbutamol compared to placebo. There were no differences in the percent changes in mean VE and Vt in the two time trials between male (VE: -0.02 (9.2)%; Vt: 0.18 (7.8)%) and female athletes (VE: 1.01 (9.5)%; Vt: 0.47 (8.4)%, p = 0.943 and p = 0.922, respectively). For the salbutamol and the placebo time trials, dyspnea and the RPEL were higher in women compared to men; however, the differences were small and were not statistically significant (see Figure 4).  Figure 4: Rating of perceived dyspnea in male and female athletes following the inhalation of salbutamol and placebo.  The dyspnea ratings from male athletes has been previously published in Koch et al.60 33  2.4 Discussion The purpose of this study was to investigate the effects of IBAs on lung function and 10-km cycling time trial performance in trained female EVH+ and EVH- athletes. The major findings of this project are fourfold: (1) there was a significant increase in FEV1 in trained female athletes after the inhalation of 400 μg of salbutamol, regardless of their EVH status; (2) despite this significant increase in lung function after the inhalation of salbutamol in female athletes, mean power output maintained over the duration of a 10-km cycling time trial was significantly decreased; (3) there were no differences in the response to 400 μg of salbutamol on lung function between male and female athletes; (4) in the salbutamol time trial, mean power output and cycling economy were significantly reduced and mean VO2 was significantly increased in female athletes but not in males.  Multiple studies on male athletes have shown a significant improvement in lung function after IBA use without an effect on athletic performance.60,62 This is the first study to report a significant increase in lung function after IBA use with a concomitant decrease in athletic performance in both EVH+ and EVH- female athletes. The reduced mean power output in the salbutamol time trial in female athletes, representing a potential ergolytic effect of salbutamol in female athletes, could be explained by an overstimulation of the adrenergic β2-system impairing athletic performance. Seventeen female athletes reported either one or a combination of the following salbutamol side effects: tremor, noticeable increase in resting HR, or a feeling of restlessness. In our previous study with men,60 who were heavier and exposed to the identical absolute dose of salbutamol (i.e. had a lower relative dose of salbutamol per kg/BM), mean VO2 was not increased in the salbutamol time trial, and side effects of salbutamol were reported in 34  only 5 out of 49 athletes. The increased relative salbutamol dose per kg/BM may also be responsible for the trend for higher HRs (especially in the first 8 km of the time trial) observed in female athletes after IBA use compared to male athletes. In contrast to this hypothesis, Sporer et al.61 did not find a dose-response relationship with regards to VO2 and HR when trained male cyclists were exposed to increasing doses (200 μg, 400 μg, and 800 μg) of IBA 15 minutes prior to a simulated 20-km cycling time trial. Further research exposing male and female athletes to the identical relative IBA dose per kg/BM is needed to investigate if differences in the relative IBA dose caused the significant increase in mean VO2, the subsequent decrease in cycling economy, and the higher prevalence of IBA-induced side effects in women of this study but not in the men who were included in our previous study.60 Interestingly, IBAs are not typically prescribed based on mass,1 which is likely why the WADA guidelines do not take dose per kg/BM into account for the permitted ß2-agonists in their prohibited list.39  One proposed ergogenic mechanism of IBAs is bronchodilation, which could improve VE during exercise and thereby increase VO2. The EVH+ and EVH- female athletes of this study demonstrated significantly increased mean VO2 during the salbutamol time trial without any effects on VE and an average decrease in performance of 4 W. Accordingly, female EVH+ and EVH- athletes in the present study showed a decreased economy during the salbutamol time trials. The increase in VO2 in female athletes is in contrast to the findings of our study in male athletes60 and other studies investigating the effects of IBAs on performance and VO2 in male athletes.61,62 At first glance, this may appear to have a negative impact on performance; however, it could be argued that a greater contribution of the aerobic system to power output could result in the sparing of anaerobic reserves, thereby providing an advantage in a sprint finish. Indeed, 35  RER was statistically lower for the 0- to 2-km interval and consistently lower after salbutamol use throughout the 10-km time trial, indicating a proportionally greater contribution of aerobic metabolism to power generation after salbutamol use. The performance enhancing impact of this finding was not demonstrated, given that there was no enhanced finish observed in the final two kilometers even with similar RPEL between the two conditions.   In a comparable study, Kalsen et al.80 recently reported a significant improvement in swim ergometer sprint performance but not in exhaustive swim performance after a supratherapeutic dose of IBAs in both trained male and female swimmers with and without AHR. The authors suggested that the much shorter ergometer test relied more on anaerobic metabolism than the exhaustive swim test, possibly explaining their divergent findings.80 In the current study, the time trials lasted between 15 minutes 50 seconds and 19 minutes 30 seconds, which represent more of an aerobic challenge than the swim ergometer test done by Kalsen et al.80 Another difference between the Kalsen et al.80 study and ours was the medication dosage. The IBA dose administered by Kalsen et al.80 (a combination of 1600 μg salbutamol, 200 μg salmeterol, and 36 μg formoterol) was substantially higher compared to the 400 μg salbutamol in our study. This supratherapeutic dose of IBAs increased respiratory muscle strength, assessed by the ratio between maximal inspiratory and maximal expiratory mouth pressure, in the asthmatic and non-asthmatic swimmers.80 Thus, in a much shorter (i.e. anaerobic) exercise challenge, an ergogenic effect of IBAs on cycling performance in female athletes might be observed.   A second proposed ergogenic mechanism of IBAs is the reduction of WOB during exercise. An IBA-induced bronchodilation could delay or prevent the onset of EFL, reduce WOB, and 36  potentially enhance athletic performance. Due to anatomical sex differences in the respiratory system, trained female athletes have been shown to be at greater risk of developing EFL, leading to an increased WOB.71,72 Furthermore, asthmatics have been shown to develop mechanical respiratory limitations during high-intensity exercise more frequently than non-asthmatics.86,87 In the present study, mean VE of the female athletes was in a range (> 90 L/min) where WOB in endurance-trained women is significantly greater than in men.71 Comparing the findings in women to our previous study in men,60 there were no differences in VE between the salbutamol and placebo time trials in women or men. Furthermore, there were no statistically significant differences in the ratings of dyspnea between the men and women. Both parameters are very indirect indications of WOB, but they seem to suggest that IBA may not significantly impact the WOB discrepancy between women and men, and between asthmatics and non-asthmatics. Further research is needed to assess the presence of mechanical limitations in an exercise bout similar to the 10-km time trials performed in this study by trained athletes. An IBA-induced increase in VE may only reduce or eliminate EFL in those athletes who are affected by mechanical limitations to ventilation, which could reduce the WOB. However, in the absence of EFL an IBA-induced increase in VE would likely increase the WOB and reduce cycling efficiency due to the increased VO2 needed to maintain a higher WOB.   A limitation of this study is the relatively low number of EVH+ athletes. However, the ergolytic effect of IBAs on 10-km time trial performance was independent of EVH status. Both EVH+ and EVH- athletes presented with a similar range and distribution of performance increases and decreases, as shown in Figure 1. Further assessment in female athletes during a race simulation with a sprint finish, including measurements such as blood lactate and peak power, is 37  recommended to investigate the effects of IBAs on athletic performance from a metabolic point of view.  2.5 Conclusions and practical implications In female EVH+ and EVH- cyclists, FEV1 was improved after the inhalation of 400 µg of salbutamol. Despite this increase in lung function, cycling performance during a 10-km time trial was decreased regardless of the athletes’ EVH status. A significantly increased mean VO2 and a decreased cycling economy after the inhalation of salbutamol in female athletes, but not in male athletes, may indicate an overstimulation of women’s adrenergic β2-system due to an increased relative salbutamol dosage compared to men (relative to BM).  Warm-up is an often neglected but important strategy in the management of EIB. In this study, all athletes performed a self-selected, 20-minute warm-up prior to the time trials. In the presence of this warm-up, there appears to be no benefit of salbutamol to cyclists with EIB in terms of symptoms or performance. If the subjects had not warmed up, the potential for a greater effect of salbutamol might exist.  The inhalation of 400 µg of salbutamol prior to exercise led to an ergolytic effect on cycling economy and induced a high incidence of side-effects such as tremor, restlessness and increased resting HR in female athletes. These potentially deleterious factors should be taken under consideration when managing female athletes with EIB.   38  Chapter 3: High-dose inhaled salbutamol does not improve 10-km cycling time trial performance  3.1 Introduction The medication of choice in the prevention and treatment of acute symptoms of EIB are IBAs.1 Due to their effects on the adrenergic nervous system and their ergogenic potential, IBAs have been added to the list of prohibited substances by WADA.39 Oral ingestion of β2-agonists has been shown to increase exercise performance of athletes in a number of studies;88-91 however, whether there is an ergogenic effect of the inhaled form remains unclear. The hypothesized cause of the differential effects of oral and intravenous vs. inhaled  β2-agonists on athletic performance,52 is the lower bioavailability of β2-agonists when taken up by inhalation, and also a more local effect in the airways. To date, solely salbutamol (maximally 1600 μg over 24 hours), formoterol (54 μg over 24 hours), and salmeterol (200 μg over 24 hours) comply with the WADA guidelines for the treatment of EIB-symptoms when delivered via inhalation.39 Studies investigating the ergogenic effects of inhaled salbutamol in therapeutic and supratherapeutic doses, ranging between 200 μg and 800 μg, did not demonstrate enhanced performance in trained athletes.60-62,92 A recent study that focussed on the effects of WADA’s maximally allowed daily doses of IBAs on athletic performance has shown that at high doses, IBAs might in fact be ergogenic.80 Following the inhalation of 1600 μg salbutamol,      200 μg salmeterol, and 36 μg formoterol, elite swimmers (VO2max: 58.7 ± 3.1 mL/kg/min) showed no improvement in a swim test to exhaustion lasting 2 minutes 45 seconds, but a shorter swim ergometer test performance of 1 minute in duration was significantly improved.80 Furthermore, maximal voluntary 39  contraction of the quadriceps muscle was significantly increased following the inhalation of the combined IBA dose.80 However, two studies by Dickinson et al. investigated the effects of acute78 and chronic79 exposures to 1600 μg of salbutamol on running performance and found no effect on running performance over a 5-km distance lasting 28 minutes 20 seconds78 and a 3-km distance lasting 16 minutes 35 seconds,79 respectively. Unfortunately, there are several limitations to these studies, such as a relatively small sample size (n = 7),78 a non-specific athletic background of the study participants (runners, soccer players, cyclists, boxers, rugby, gymnastic and tennis)79 and relatively low cardiorespiratory fitness levels.78,79 Thus, the applicability of the Dickinson et al.78,79 non-ergogenic findings of high-dose salbutamol exposures to trained endurance athletes is limited. Thus, although at low and moderate doses, there does not appear to be an ergogenic effect for salbutamol,60,92 there is a potential for high doses (1600 μg) to be ergogenic in trained athletes. Furthermore, it is unclear whether high-dose inhaled salbutamol induces identical bronchodilatory, and possibly ergogenic, effects in athletes with mild EIB and healthy athletes without EIB, or if there are differences.  One other factor that has not yet been analyzed is the impact of relative IBA dose (i.e. μg per kg/BM) on athletic performance. The guidelines for the maximally allowed daily doses of IBAs are not normalized by BM, which means that lighter athletes may possibly benefit from a considerably greater IBA dose per kg/BM compared to heavier athletes. The purpose of this study was to investigate the effects of high-dose inhaled salbutamol on 10-km cycling time trial performance in trained cyclists and triathletes with and without EIB. We hypothesized that in trained male athletes, salbutamol would improve time trial performance, and the benefits would be more pronounced in those individuals receiving a higher relative dose. 40  3.2 Methods 3.2.1 Study participants Twenty trained male cyclists and triathletes were included in this study. Participants had no history of cardiopulmonary disease (except for controlled EIB) and reached a VO2max ≥             60 mL/kg/min or 5 L/min in the graded exercise test on the screening day. Study participants were recruited though study posters that were circulated in electronic newsletters of local cycling and triathlon clubs, word-of-mouth advertising, and posters hung on message boards of local bike shops. Written informed consent was obtained from all subjects prior to data collection. The study was performed in accordance with the Helsinki declaration and with approval from the University of British Columbia Ethics Board.  3.2.2 Experimental protocol The study was designed using a double-blind, placebo-controlled, randomized cross-over protocol. Each athlete visited the laboratory on three different occasions. The first visit served to screen for EIB and to assess cardiovascular fitness. Furthermore, participants were familiarized with spirometric testing and the 10-km time trial distance. On the following two test days, athletes performed one simulated 10-km time trial on each test day, separated by a minimum of  three and a maximum of 14 days following the inhalation of salbutamol and placebo. For every athlete, the two time trial visits took place at the same time of the day. Athletes were asked to abstain from β2-agonists for at least 12 hours prior to arrival but were allowed to continue corticosteroid treatments. Athletes did not exercise prior to coming to the laboratory on testing days to avoid exercise-induced bronchodilation or EIB prior to data collection. Additionally, athletes refrained from caffeinated beverages on testing days. All tests were performed in the 41  same laboratory, in the same season with an average ambient temperature of approximately 22°C, and an average humidity of approximately 72%. 3.2.3 Screening visit Bronchial hyperresponsiveness was assessed using the EVH test.46 Lung function was measured with spirometry84 (TrueOne 2400; ParvoMedics, Sandy, UT, USA), and the highest FEV1 from three maneuvers was used as baseline. Following the guidelines of the American Thoracic Society (ATS), spirometry maneuvers were repeated if FVC or FEV1 did not fall within 150 mL of each other.84 Athletes then hyperventilated dry gas (5% CO2) for six minutes and repeated spirometry 3, 5, 10, 15, and 20 minutes post-hyperventilation. A decrease in FEV1 ≥ 10% in two consecutive measurements relative to baseline was classified as EVH+.46 Cardiorespiratory fitness was determined by VO2max during a GXT on a cycle ergometer (Velotron Dynafit Pro, RacerMate Inc., Seattle, WA, USA). The test began at 100 W, and work rate increased by 0.5 W/second until cycling cadence was < 60 rpm.  After the GXT, participants were told to continue cycling until they felt sufficiently recovered to perform a practice 10-km time trial. The purpose of the practice cycling time trial was to familiarize athletes with the virtual gearing system of the bicycle ergometer and to minimize learning effects between the time trials performed on test days one and two. Athletes performed the practice time trials wearing a facemask (Hans Rudolph Inc., 7450V2 Mask; Shawnee, KS, USA) connected to a metabolic cart (TrueOne 2400; ParvoMedics, Sandy, UT, USA).  42  3.2.4 Time trial visits Athletes performed spirometry to determine the effect of each treatment on lung function, specifically FEV1, FVC and FEV1/FVC, immediately before, and 15 minutes following the inhalation of salbutamol or placebo (Apotex Inc., Toronto, Canada). Upon completion of the second spirometry test, athletes began a predetermined warm-up of 15 minutes. The warm-up consisted of two minutes cycling at 30% of each athlete’s maximal cycling resistance reached during the GXT on the screening day. Over the period of one minute, the intensity was then increased to 50% of maximal resistance for two minutes. Finally, five sprints at 90% of the maximal cycling resistance for 30 seconds with 90 seconds at 50% in between completed the warm-up. As per the practice time trial, subjects wore a facemask connected to a metabolic cart during the 10-km time trials. A time trial course (3D Cycling, RacerMate Inc., Seattle, WA, USA) was displayed on a screen, with distance, cadence, and gearing information displayed. Athletes rated dyspnea and RPEL on a 0 - 10 Borg-scale85 every two kilometers. Immediately upon completion of the time trials, FEV1 was reassessed by traditional spirometry. Whenever possible, we aimed to perform three spirometry maneuvers; however, due to high levels of breathlessness, some athletes were only able to perform two repeatable maneuvers. The main outcome variable was mean power output over the duration of the 10-km time trial. Secondary outcome variables were VO2, RER, peripheral capillary oxygen saturation (SpO2), HR, VE, Vt, RR, dyspnea, and RPEL. To assess for a possible time effect of salbutamol on the outcome variables, these parameters were averaged for each 2-km interval.  43  3.2.5 Statistical analyses All data are presented as means including standard deviations. The effect of EVH status on anthropometrics, spirometric, and baseline fitness parameters was assessed using the independent samples t-test. The effects of drug treatment and EVH status on the assessed performance parameters were tested with ANCOVA tests. Post-hoc analyses were performed using Tukey’s HSD test. To compare the effects of salbutamol dose per kg/BM, salbutamol dose of 1600 μg was normalised to each athlete’s BM. The relationship between dose per kg/BM and cardiorespiratory parameters (specifically, the percent change of every assessed cardiorespiratory and performance parameter between the means of the salbutamol and the placebo time trials) was investigated using the Pearson product-moment correlation coefficient. Preliminary analyses were performed to ensure that there was no violation of the assumptions of normality, linearity, and homoscedasticity. Statistical analyses were completed using SPSS (IBM, Version 22.0, Armonk, NY, USA), and statistical significance was accepted when p < 0.05.  3.3 Results 3.3.1 Participant characteristics  Eight of the 20 included athletes were classified as EVH+. The mean decrease in FEV1 following the 6 minute hyperventilation phase of the EVH test was 13.3 (3.3)% in EVH+ athletes compared to 2.3 (4.9)% in EVH- athletes (p < 0.001). Of the eight EVH+ athletes, only three had a history of asthma. One EVH+ athlete was treating his asthma symptoms with daily ICS and IBAs on an as-needed basis. The remaining two EVH+ athletes with a prior diagnosis of asthma treated their symptoms solely with IBAs on an as-needed basis. There were no differences in anthropometrics, cycling experience, or fitness levels between EVH+ and EVH- athletes; 44  however, the ratio between FEV1/FVC was significantly smaller in EVH+ athletes 73 (7)% compared to EVH- athletes (80 (7)%, p = 0.042). All anthropometric, fitness, and spirometric parameters are summarized in Table 3.   45  Table 3: Anthropometric, fitness, and spirometric parameters in male EVH- and EVH+ athletes. Parameter Total (n = 20) EVH– (n = 12) EVH+ (n = 8) Mean (SD) Range Mean (SD) Range Mean (SD) Range Age (years) 20 (6) 20 – 45 31 (7) 22 – 45 29 (5) 20 – 37 Height (cm) 180 (5) 170 – 188 180 (6) 170 – 188 181 (4) 173 – 185 Mass (kg)  75 (7) 59 – 90 75 (7) 59 – 88 76 (7) 67 – 90 Cycling experience (years) 5 (5) 1 – 14 4 (1) 1 – 14 5 (2) 3 – 10 Cycling volume (hrs/week) 11 (6) 3 – 23 11 (5) 3 – 19 12 (6) 6 – 23 FVC (L) 6.5 (1.0) 4.1 – 9.0 6.5 (1.3) 4.1 – 9.0 6.5 (0.5) 5.5 – 7.1 FVC predicted (%) 117 (14) 79 – 149 117 (18) 79 – 149 116 (5) 111 – 113 FEV1 (L) 5.0 (0.9) 3.8 – 7.5 5.1 (1.0) 43.8 – 7.6 4.9 (0.6) 4.0 – 5.6 FEV1 predicted (%) 112 (13) 89 – 154 115 (15) 93 – 154 107 (8) 89 – 114 FEV1/FVC (%)a 77 (7) 64– 92 80 (7) 71 – 92 73 (7) 63 – 81 FEV1/FVC predicted (%) 94 (9) 77 – 119 98 (9) 85 – 119 90 (7) 77 – 99 Δ Max FEV1 (%)b 7 (7) -7 – 20 2 (5) -7 – 8 13 (3) 10 – 20 VO2max (mL/kg/min) 64.2 (5.3) 56.6 – 77.7 64.8 (6.0) 56.7 – 77.7 63.2 (4.1) 56.6 – 70.5 Maximal power (W) 431 (31) 373 – 502 435 (34) 379 – 502 425 (29) 373 – 464  FVC, forced vital capacity; FEV1, forced expiratory volume in 1 second; Δ Max FEV1, percent decrease in FEV1 following the eucapnic voluntary hyperpnea test, VO2max, maximal oxygen consumption.        a FEV1/FVC was significantly greater at baseline in EVH- athletes compared to EHV+ athletes (p = 0.042). b The decrease in FEV1 following the EVH challenge was significantly greater in EVH+ compared with EVH- athletes (p < 0.001).  46  3.3.2 The effect of 1600 µg salbutamol on lung function and athletic performance On the time trial visits, FEV1 improved by 6.4 (4.9)% following the salbutamol treatment compared to 1.0 (4.4)% following the placebo treatment (p < 0.001). There was no statistically significant difference in the bronchodilatory effect of inhaled salbutamol between EVH+           (Δ FEV1 = 5.8 (4.8)%) and EVH- athletes (Δ FEV1 = 6.7 (5.1)%, p = 0.691). Despite this increase in FEV1, time trial performance was not improved following the inhalation of 1600 μg of salbutamol (see Figure 5), regardless of athletes’ dose of inhaled salbutamol per kg/BM (see Table 4) and EVH status. In Table 5, the cardiorespiratory parameters are summarized by EVH status and 2-km bouts. The mean duration of the 10-km cycling time trial was 15 minutes         34 seconds for the salbutamol and 15 minutes 39 seconds for the placebo time trial (p = 0.623). The inhaled salbutamol dose per kg/BM did correlate with the percent change in FEV1 following drug treatment (p = 0.004), but there were no correlations for any of the assessed performance and metabolic parameters between the salbutamol or placebo time trials and inhaled dose (see Table 4).   Figure 5: Athletic performance after salbutamol and placebo inhalation in EVH- athletes (A) and EVH+ athletes (B). The dashed line represents the mean performance for EVH+ and EVH- athletes.47  Table 4: Correlations between the relative dose of inhaled salbutamol (µg per kg/BM) and spirometric as well as performance parameters. Parameters Forced expiratory volume (FEV1)  L Mean power output   (Power)  W Minute ventilation  (VE)  L/min Oxygen consumption  (VO2)  mL/kg/min Respiratory exchange ratio (RER) Heart rate   (HR)  bpm Oxygen saturation  (SpO2)  % Pearson Correlation -.612 .182 -.003 .187 -.134 -.190 -.114 Sig.           (2-tailed) 0.004* 0.443 0.989 0.429 0.573 0.422 0.633 * The percent change in FEV1 was significantly correlated with the inhaled dose of salbutamol per kilogram of body mass.   48  The FEV1 assessed prior to the inhalation of either placebo or salbutamol did not indicate bronchoconstriction experienced by EVH+ athletes. The percent predicted FEV1 values of the EVH+ athletes were at 108% (ranging from 98% - 118%), and 102% (ranging from 89% - 110%) prior to the inhalation of salbutamol and placebo, respectively. The spirometry that was performed immediately upon completion of every time trial did not indicate bronchoconstriction, suggesting no asthma attack as a result of the cycling time trials following salbutamol or placebo treatment in EVH+ or EVH- athletes. After the placebo time trials, two EVH- athletes’ FEV1 was 6% and 1% lower, respectively, compared to the FEV1 taken prior to the placebo inhalation, before the warm-up. After the salbutamol time trials, none of the EVH- athletes presented with a lower FEV1 compared to the FEV1 assessed prior to the salbutamol treatment. Among the EVH+ athletes, the FEV1 of one athlete dropped by 4% following the placebo time trial. The FEV1 of another EVH+ athlete was decreased by 6% following the salbutamol time trial.  Mean VE (p = 0.034), RR (p = 0.014), and HR (p = 0.010) were significantly increased in the salbutamol time trial compared to the placebo time trial (see Table 5). The RER and SpO2 were not affected by salbutamol. The rating of perceived dyspnea was not altered by inhaled salbutamol; however, RPEL was significantly increased in the salbutamol time trial compared to the placebo time trial in EVH+ and EVH- athletes (p = 0.039, see Figure 6).    49  Table 5: Mean and standard deviations for the 2-km time trial interval analysis after inhalation of 1600 µg salbutamol or placebo. Parameter (Units) Treatment condition and EVH status Distance 0-2km M (SD) 2-4km M (SD) 4-6km M (SD) 6-8km M (SD) 8-10km M (SD) Power  (Watts) Salbutamol  Total EVH- EVH+  314 (43) 321 (51) 305 (31)  301 (38) 308 (43) 290 (28)  290 (28) 293 (27) 285 (31)  287 (24) 290 (21) 282 (28)  316 (33) 319 (37) 313 (26)  Placebo  Total EVH- EVH+  312 (37) 318 (39) 304 (35)  306 (40) 317 (48) 291 (23)  295 (31) 303 (37) 283 (15)  292 (29) 298 (36) 283 (13)  316 (35) 319 (39) 312 (30) VO2  (mL/kg/min) Salbutamol Total  EVH- EVH+  48.7 (4.8) 49.4 (5.4) 47.6 (3.8)  54.8 (5.4) 56.1 (5.7) 52.7 (4.4)  54.0 (4.8) 54.9 (4.6) 52.7 (5.1)  53.6 (4.7) 54.4 (4.3) 52.3 (5.1)  55.1 (4.5) 55.7 (4.2) 54.1 (5.1) Placebo  Total EVH- EVH+  47.2 (4.7) 47.3 (5.4) 47.1 (3.9)  53.6 (6.2) 54.5 (7.2) 52.2 (4.3)  53.6 (5.3) 54.1 (6.1) 52.9 (4.1)  53.6 (4.8) 54.1 (5.4) 52.8 (3.8)  54.9 (4.8) 55.2 (4.6) 54.5 (5.6) METS  kcal/kg/hr Salbutamol  Total EVH- EVH+  13. 9 (1.4) 14.1 (1.5) 13.6 (1.1)  15.6 (1.5) 16.0 (1.5) 15.1 (1.3)  15.4 (1.4) 15.7 (1.3) 15.1 (1.5)  15.3 (1.3) 15.5 (1.2) 14.9 (1.5)  15.7 (1.3) 15.9 (1.2) 15.5 (1.4) Placebo Total  EVH- EVH+  13.5 (1.4) 13.5 (1.5) 13.4 (1.1)  15.3 (1.4) 15.7 (2.1) 14.9 (1.3)  15.3 (1.5) 15.5 (1.7) 15.1 (1.2)  15.3 (1.4) 15.4 (1.5) 15.1 (1.1)  15.7 (1.4) 15.8 (1.3) 15.6 (1.4) VE  (L/min) Salbutamol Total  EVH- EVH+  108 (16)a 110 (21) 105 (6)  129 (23)a 133 (28) 124 (12)  133 (24)a 135 (28) 129 (19)  134 (24)a 136 (27) 131 (22)  144 (23)a 146 (26) 141 (20)  Placebo  Total EVH- EVH+  101 (14) 99 (17) 103 (10)  120 (22) 120 (27) 120 (13)  126 (24) 125 (29) 127 (16)  129 (25) 129 (30) 129 (17)  138 (28) 138 (32) 139 (22) 50  Parameter (Units) Treatment condition and EVH status Distance 0-2km M (SD) 2-4km M (SD) 4-6km M (SD) 6-8km M (SD) 8-10km M (SD) RR  (breaths/ minute) Salbutamol Total  EVH- EVH+  35 (6)b 34 (7) 37 (4)  41 (8)b 40 (10) 43 (4)  46 (11)b 44 (12) 47 (8)  46 (11)b 44 (12) 48 (10)  50 (11)b 48 (12) 52 (9) Placebo  Total EVH- EVH+  34 (7) 32 (7) 37 (3)  39 (9) 37 (11) 42 (5)  42 (10) 37 (11) 42 (5)  44 (11) 42 (13) 48 (9)  48 (12) 46 (14) 52 (9) Vt  (L) Salbutamol  Total EVH- EVH+  3.1 (0.5) 3.3 (0.6)c 2.8 (0.2)  3.2 (0.5) 3.5 (0.6)c 2.9 (0.2)  3.1 (0.5) 3.3 (0.5)c 2.8 (0.2)  3.0 (0.5) 3.2 (0.5)c 2.7 (0.2)  3.0 (0.5) 3.1 (0.5)c 2.7 (0.2)  Placebo  Total EVH- EVH+  3.1 (0.6) 3.2 (0.7)c 2.8 (0.2)  3.2 (0.6) 3.4 (0.7)c 2.9 (0.2)  3.09 (0.6) 3.29 (0.7)c 2.78 (0.2)  3.0 (0.6) 3.2 (0.7)c 2.7 (0.3)  3.0 (0.6) 3.1 (0.6)c 2.7 (0.3) HR  (beats/ minute) Salbutamol Total  EVH- EVH+  165 (6)d 166 (7) 165 (4)  174 (5)d 175 (6) 173 (3)  176 (6)d 176 (7) 175 (6)  177 (6)d 177 (7) 176 (4)  180 (6)d 180 (8) 180 (5)  Placebo  Total EVH- EVH+  157 (9) 155 (10) 158 (8)  169 (10) 169 (11) 169 (8)  172 (11) 172 (13) 173 (8)  173 (13) 171 (16) 175 (8)  177 (12) 175 (13) 179 (9) VO2, oxygen consumption; METS, metabolic equivalent of task; VE, minute ventilation; RR, respiratory rate; Vt, tidal volume, HR, heart rate; EVH+, athletes with a positive eucapnic voluntary hyperpnea test; EVH- athletes with a negative eucapnic voluntary hyperpnea test.   a VE was significantly greater after inhalation of salbutamol compared with placebo (p = 0.030). b RR was significantly greater after inhalation of salbutamol compared with placebo (p = 0.013). c Vt was significantly greater in EVH- athletes compared with that in EVH+ athletes regardless of the drug treatment (p = 0.044). d HR was significantly greater after inhalation of salbutamol compared with placebo (p = 0.009).    51  Mean Vt was significantly greater in EVH- athletes compared to EVH+ athletes in both time trials (p = 0.044). Respiratory rate was greater in EVH+ athletes compared to EVH- athletes throughout both time trials; however, this difference did not reach statistical significance. Mean VE was greater in EVH- athletes compared to EVH+ athletes in both time trials, but again, this did not reach statistical significance (p = 0.056).   Figure 6: Ratings of perceived dyspnea (A) and perceived leg fatigue (B) displayed as means and standard deviations. * indicates that a significant main effect of salbutamol. Post-hoc testing using Tukey’s HSD identified the 2-km intervals at which statistically significant differences between the salbutamol and placebo condition existed between the two means.   3.4 Discussion The main findings of this study are fourfold: Primarily, the inhalation of WADA’s maximal allowed daily dose of 1600 μg of salbutamol did not lead to an enhanced 10-km time trial performance in trained male cyclists. Secondly, there was no difference with respect to bronchodilatory effect or 10-km cycling time trial performance after the inhalation of 1600 μg of 52  salbutamol between EVH+ and EVH- athletes. Thirdly, the dose of inhaled salbutamol per kg/BM did not affect cycling performance. Athletes with a lower BM did not benefit from the greater relative dose compared to heavier athletes. Lastly, the acute dose of 1600 μg of salbutamol led to a number of side effects, such as increases in HR, RR, VE, and RPEL. In a previous study, we exposed trained male EVH+ and EVH- cyclists to 400 μg of inhaled salbutamol and observed an increase in FEV1 of 6.1 (4.7)%.60 Even though athletes were exposed to a four-fold greater dose of salbutamol in the present study, the magnitude of the bronchodilatory effect (6.4 (4.9)%) was comparable and remained below a clinically significant bronchodilation of 12%.62 The absence of a linear dose-response relationship with regards to IBA-induced bronchodilation is in accordance with the findings by Dickinson et al.78 who exposed athletes to 800 μg and 1600 μg of inhaled salbutamol in a repeated-measures study design. After the exposure to either dose, mean FEV1 increased from 4.6 (0.9) L at baseline to 4.7 (0.9) L after either salbutamol dose. This bronchodilatory effect of only 2% was considerably lower than the one demonstrated in the present study; furthermore, Dickinson et al.78 did not take inhaled dose per kg/BM into consideration. The present study is the first to show a correlation between the inhaled dose of salbutamol per kg/BM and the induced percent increase in FEV1.   As mentioned previously, one mechanism that was thought to explain a potential ergogenic effect of IBAs was based on its bronchodilatory properties and the potential to increase VE and VO2. In the present study, salbutamol induced a bronchodilatory effect of 6.4%, which led to a significant increase in VE throughout the duration of the entire salbutamol 10-km time trial; however, this increase in VE did not lead to an improved performance. As previously mentioned, 53  Dickinson et al.78 report a bronchodilatory effect of only 2% following the inhalation of the identical dose of salbutamol as in the present study, but there were no significant increases in VE, HR, or VO2 in runners over a 5-km distance. In the present study, the increase in VE was primarily driven by an increase in RR rather than Vt. This could indicate a secondary stimulation of the adrenergic nervous system as opposed to dilated airways facilitating the respiratory work during high-intensity exercise by, for example, increasing the amount of linear airflow and decreasing the proportion of turbulent flow. This hypothesis is supported by identical ratings of perceived dyspnea between the salbutamol and placebo time trials. Another sign indicative of an overstimulation of the adrenergic nervous system was the significantly increased HR in the salbutamol time trial. Shortly after the completion of the inhalation, all 20 athletes reported one or several common side effects of β2-agonist exposure such as tremor, tachycardia, or restlessness.   In the present study, all athletes performed a predetermined warm-up that included five            30- second sprints at 90% of every athlete’s maximal cycling resistance reached during the GXT on the screening day. As per Stickland et al.,49 a warm-up that includes short-duration, high-intensity intervals ideally prepares asthmatic athletes for upcoming exercise bouts. The greatest drop in FEV1 immediately upon completion of the placebo time trial was 6% in one EVH- athlete and 6% in an EVH+ athlete after completing the salbutamol time trial. Since a drop in FEV1 of    ≥ 10% is defined as an asthma attack,these findings could indicate that the predetermined warm-up indeed optimally prepared athletes for an exercise bout, such as the 10-km time trial, possibly by inducing a refractory period in the EVH+ athletes. It is possible that a refractory period 54  following the warm-up in the placebo time trial could, in part, explain the lack of difference in performance with respect to the salbutamol time trial; however, the predetermined warm-up in this study is comparable to the warm-up athletes perform prior to a race and simulates real-competition situations in which athletes on IBAs appear to be more successful compared to athletes who do not use IBAs.15 With the current study design, we did not assess athletes in the absence of a warm-up, so cannot make conclusions about the actual efficacy of the warm-up in maintaining lung function during the bout of exercise. Furthermore, athletes should not take these findings to conclude that IBAs are no longer necessary for the treatment of EIB. Other reasons that could account for the lack of difference between the salbutamol and the placebo time trials could be that the EVH challenge was too sensitive and the exercise environment in the laboratory possibly not provocative enough to induce EIB in the placebo time trials. It is also possible that only athletes with a more severe drop in FEV1 following the EVH challenge benefit from high-dose salbutamol with respect to athletic performance and that the included cyclists in our study (drop in FEV1 following EVH challenge = 13%) did not exceed this threshold.   The trained cyclists included in this study reported greater RPEL in the salbutamol condition than in the placebo condition. This finding of an increased perceived exertion was also recently demonstrated by Dickinson et al.78 in runners who rated their perceived exertion at kilometers two, three, and four significantly greater following salbutamol administration than during the placebo 5-km run. The consistency of this finding is intriguing. One of the known adverse effects of salbutamol is muscle cramping, and with higher doses, elevations in serum creatine kinase levels (indicating muscle damage) have been demonstrated.93-95 These higher RPEL values in both studies could result from a similar mechanism as the cramping. One might expect that these 55  increased RPEL ratings might be ergolytic for the athletes; however, in neither study does the increase in RPEL lead to a significant effect on athletic performance. Salbutamol-mediated muscle cramping is associated with high doses of salbutamol and oral administration of the medication.93,94 Similarly, the phenomenon of an increased RPEL during exercise appears to be unique to the high-dose IBA administration. In our previous work, using the same methodology and population with moderate doses of salbutamol,92 there was no effect of salbutamol on RPEL whatsoever.   This study does not suggest that the relative dose of salbutamol per kg/BM has an ergogenic impact on athletic performance, even though the BM of the included athletes ranged between    67 kg and 90 kg. This variation corresponds with salbutamol doses ranging from 17.8 μg         per kg/BM to 23.7 μg per kg/BM or a maximal difference of 33% between the lowest and highest dose of inhaled salbutamol per kg/BM. Despite this considerable difference in relative dose, no effect of relative dose was seen.  The investigation of the ergogenic potential of IBAs has recently focused on the duration and metabolic properties of the assessed exercise bouts. The present study is the first to investigate the effects of 1600 μg of salbutamol on cycling performance lasting ∼15 minutes 30 seconds. Athletes maintained an average RER of 1.02, indicating an exercise intensity close to the anaerobic threshold. Most studies that investigated the ergogenic effects of IBAs in exercise protocols lasting longer than two minutes, even after acute or chronic supramaximal IBA treatment, did not show an enhanced athletic performance.78 Following the inhalation of the maximal dose of three β2-agonists, swimmers significantly improved their performance in a 56  swimming ergometer sprint test that lasted just under one minute.80 Hostrup et al.96 found a significant increase in peak power during the first of three 30-second Wingate tests after a single acute dose of salbutamol in cyclists and an improved peak power in the first and second of three Wingate tests following a two-week treatment of salbutamol; however, these athletes were treated with 8 mg of oral salbutamol as opposed to inhaled salbutamol in the present study. These findings suggest that future research should focus on the investigation of the ergogenic potential of high-dose IBAs in exercise protocol shorter than 2 minutes with very high exercise intensities. One limitation of this study was the difficulty of blinding the athletes and researchers to the treatment condition due to the high prevalence of experienced side effects. Consequently, researchers standardized the verbal encouragement given during the spirometry and exercise testing. Furthermore, this study focused on the effects of high-dose salbutamol in male athletes only. To expand the range of exposed doses of inhaled salbutamol per kg/BM further, and to investigate differences between men and women, this research should be extended to a female athlete population. Previous research in our group on female cyclists and triathletes showed that the standard 400 μg of inhaled salbutamol did not improve 10-km cycling performance.92  3.5 Conclusion Following the WADA guidelines39 with regards to the use of IBAs, the inhalation of the maximally allowed daily dose of salbutamol of 1600 μg did not improve 10-km time trial performance in trained male cyclists, regardless of EVH status and relative dose per kg/BM. Significant increases in HR, VE, RR, and RPEL appear to be indicative of an overstimulation of the adrenergic nervous system without leading to enhanced athletic performance in an exercise 57  protocol of 15 minutes 30 seconds in duration and an intensity at the aerobic-anaerobic threshold. 58  Chapter 4: Evaluation of the diagnostic criteria of the eucapnic voluntary hyperpnea test in trained athletes  4.1 Introduction Mucosal dehydration caused by evaporation and mucosal cooling with subsequent hyperaemia are the stimuli thought to cause EIB.35 The reported prevalence of EIB ranges considerably in elite athletes (30% - 70%)97 and recreationally active populations (13% - 50%).40,98 One reason for the wide range in EIB prevalence is the varying degree by which the investigated populations are affected by the main risk factors in the pathogenesis of EIB, such as: sporting discipline,15 volume and intensity of physical activity,97 age and duration of athletic career,98,99 air quality,23 and environmental conditions.100 Another explanation for the wide range in EIB prevalence is the diversity in diagnostic approach. Respiratory symptoms are poor predictors of EIB,101 which makes a symptom-based approach for diagnosis inadequate.102 Generally, EIB is diagnosed on the basis of an abnormal reduction in FEV1. An abnormal reduction is typically defined as the mean plus two standard deviations of the decrease in FEV1 documented in healthy subjects after exposure to a bronchoconstriction stimulus.35 Currently, there is no gold standard for the diagnosis of EIB; however, the International Olympic Committee (IOC) considers the EVH test the best available laboratory-based challenge.45 The EVH test requires the hyperventilation of compressed gas (including 5% CO2) for six minutes, followed by spirometry.46 Spirometry is performed at baseline and repeated in duplicate at multiple time points post-hyperventilation. An FI ≥ 10% is considered a positive test, thus diagnosing the test taker with EIB+.   59  The EVH challenge was first described as a diagnostic test for EIB in the U.S. Army in the mid-1980s.43,103 Over the years, the EVH challenge has been continuously adjusted in order to minimize the potential for misdiagnosis. To this date, questions concerning the test protocol and interpretation are the reason for ongoing research. Some of the changes within the EVH test that have been discussed or implemented since its introduction are:   (1) The calculation of the FI is based on two consecutive values14,104 with a decrease ≥ 10% rather than just one FEV1 value.42,44,46 Two consecutive measurements are thought to minimize the risk of a low reading resulting from a single poor effort or technique error instead of a sustained reduction in lung function.14   (2) The normalization of the FI by the VEEVH-achieved relative to VEEVH-target.42,44 High VEs are thought to result in greater evaporative water loss.14,42 Thermal (airway cooling with subsequent hyperaemia) and osmotic (increase in osmolarity due to decrease in airway surface liquid) effects on evaporative water loss lead to the release of mediators that eventually induce bronchoconstriction.   (3) A shift of the cutoff for an EVH+ test indicative of EIB+ from 10% to 15%.47 Recently, Price et al.47 suggested a FI ≥ 15% may be a more appropriate cutoff for an EVH+ diagnosis because a fall in FEV1 ≥ 10% may be encountered in approximately 20% of asymptomatic, healthy athletes, which may represent a “variation of the normative airway response” following EVH. Due to a lack of data on inflammatory markers, it is unclear if this intermediate group is truly healthy.105  60  The number of consecutive post-challenge values included in the calculation of the FI, the intensity of the bronchoconstriction stimulus, and the threshold used to distinguish between an EVH+ and an EVH- test could affect the diagnosis, and subsequently, the treatment and training strategy, particularly in those athletes responding to an EVH challenge with an FI of just above, or just below 10%. To our knowledge, none of the above outlined suggestions have been supported or disputed by robust data, thus necessitating this retrospective analysis. Several studies show that EIB is both over- and underdiagnosed.106-108 Failure to properly identify EIB can lead to poorly managed symptoms, an impairment of athletic performance, and in extreme cases, it can act as a barrier to engagement in physical activity, thus impairing quality of life.109,110 Mistakenly diagnosing and treating an individual as EIB+ when they have another condition can lead to unnecessary and ineffective medication, without relief of symptoms, and potential adverse effects in response to the medication. The aim of this retrospective analysis was to evaluate four calculation methods of the FI that have been used since the introduction of the EVH challenge. We compared the FIs using these varying criteria to changes in FEV1 following a short, intense exercise bout to get an estimate of ecological validity. Additionally, we evaluated the intensity of the bronchoconstriction stimulus generated by the EVH challenge by comparing the VEs generated in the EVH challenge to the VEs achieved in an intense, short exercise bout and in the GXT by trained male and female athletes.    61  4.2 Methods 4.2.1 Preliminary remarks This study was performed as a retrospective analysis of the data collected for the studies outlined in Chapters 2 and 3. The EVH challenges used to screen athletes for EIB in Chapters 2 and 3 served for the calculation of the FIs using four previously published methods, which are described in section 4.2.5. For every athlete, the four FIs were compared to the change in FEV1 induced by the time trial following the inhalation of placebo. Lastly, VEs generated in the GXT (VEGXT) of the assessment days and the VEs during the final two kilometers of the placebo time trials (VETT) were used to evaluate the intensity of the bronchoconstriction stimulus of the EVH challenge. A total of 39 athletes (18 males and 19 females) of the 27 screened female athletes in Chapter 2 and the 20 screened male athletes in Chapter 3 were included in this analysis. Spirometric data in duplicate at every post-hyperpnea time point for the EVH challenge and spirometry in duplicate following the placebo time trial served as inclusion criteria for this retrospective analysis. Six athletes from Chapters 2 and 3 were excluded based on only one spirometry maneuver at one of the five post-hyperpnea time points in the EVH challenge, and four athletes were unable to provide a reproducible FEV1 assessment following the time trial.   4.2.2 Study population As part of the two previously published studies, we screened 37 trained male111 and female92 athletes for EIB using the EVH test. All athletes were non-smokers, not pregnant, and free from cardiovascular and metabolic disease. As outlined in Chapter 2, women were tested at random points throughout their menstrual cycle, and the use of oral contraceptives was not an exclusion criterion. Approval for this study was obtained by the University of British Columbia Clinical 62  Research Ethics Board in accordance with the Helsinki declaration and all study participants provided written, informed consent.  4.2.3 Experimental protocol The data for this analysis were obtained from two visits to the laboratory: a screening visit and a time trial visit. On both test days, participants proceeded with the testing battery if their baseline FEV1 was > 80% of predicted value. The washout period between these two visits was a minimum of 72 hours and a maximum of 14 days. On the screening visit, participants completed an EVH challenge followed by a GXT test on a cycle ergometer. On the time trial visit, athletes completed a 10-km time trial on a cycle ergometer following a warm-up.   4.2.4 Screening visit On the screening visit, the EVH challenge was conducted before the GXT, and the two tests were separated by a break that was individually determined by the athletes. Spirometry was performed prior to the start of the GXT to ensure FEV1 had returned to baseline (FEV1 within 150 mL or 5% of baseline). Athletes with a FI ≥ 10% were given 200 μg of salbutamol to reverse the   EVH-induced bronchoconstriction (N = 8 men and 6 women). Athletes who were regularly taking asthma medication were asked to withhold from IBAs for 12 hours prior to testing but were permitted to continue their ICS treatment. Participants did not exercise on the day of testing to avoid exercise-induced bronchodilation or EIB and abstained from caffeinated beverages. All tests took place in the same laboratory and in the same season with an average ambient temperature of approximately 22° C and an average humidity of approximately 72%.  63  4.2.5 EVH challenge and calculation of fall indices The EVH challenge was performed following the guidelines provided by Anderson et al.46 Baseline lung function was determined using spirometry (TrueOne 2400; ParvoMedics, Sandy, UT, USA).84 The highest FEV1 from three maneuvers, which fell within 150mL or 5% of each other, was used as baseline. For the EVH challenge, VEEVH-target was calculated as 30 x baseline FEV1. Athletes breathed dry gas for six minutes wearing a nose clip, and repeated spirometry in duplicate 3, 5, 10, 15, and 20 minutes after completion of the hyperventilation. Real-time feedback of VE was provided as 10-second averages to ensure that the target level was maintained. If VEEVH-target was not met, the participants were verbally coached and encouraged to increase RR or Vt based on the test administrator’s observations. Typically, the first minute of the EVH challenge was used to adjust RR and Vt in order to meet VEEVH-target; therefore,   VEEVH-achieved was calculated as the mean VE of the final five minutes of EVH challenge.   The FI is calculated as:112 (Highest FEV1 value pre-hyperpnea) – (lowest FEV1 value post hyperpnea) x 100 _____________________________________________________________________ (Highest FEV1 value pre-hyperpnea)   To investigate how different strategies in the determination of the lowest FEV1 value post-hyperpnea affect the outcome of the EVH challenge, we used the following four approaches:   64  Fall Index A (FIA) was calculated following the guidelines provided by the Joint Task Force on Practice Parameters on EIB.14 The higher of two FEV1 values for each time point following hyperventilation was used for the calculation of the FI.14 If the decrease in FEV1 ≥ 10% was at two consecutive time points, a test was considered EVH+. The FI was reported using the higher FEV1 value of the two.  Fall Index B (FIB) was calculated by looking at each post-challenge FEV1 value individually, as previously used in our laboratory.111 If the decrease in FEV1 was greater than or equal to 10% in two consecutive measurements, either between two time points or within duplicate measurements at one single time point, a test was considered EVH+. The FI was reported as the higher FEV1 value of the two.   Fall Index C (FIC) was based on recommendations provided by Hurwitz et al.42 and Anderson et al.46 The higher FEV1 value of the two spirometry maneuvers at every time point post-hyperpnea was considered for the FI calculation.42 Of the five post-challenge values, the one showing the greatest decline in FEV1 from baseline was taken for the calculation of FIC. A test was considered positive if the decrease in FEV1 was greater than or equal to 10 %.  Fall Index D (FID): Following Hurwitz et al.,42 FI was normalized to VEEVH-achieved using the following equation: FID = (FEV1baseline – FEV1post-EVH) • (30 / VEEVH−achievedFEV1baseline ) In this equation, FEV1baseline – FEV1post-EVH was determined identically to FIC to adhere to the protocol outlined by Hurwitz et al.42 65  4.2.6 Maximal exercise test A GXT was performed to assess maximal VE on a sport specific test. Parameters including RR, Vt, and VEGXT were assessed as the highest 15-second average in the final minute of the GXT on a cycle ergometer (Velotron Dynafit Pro; RacerMate, Inc., Seattle, WA). The test began at 0 W and 100 W for women and men, respectively, and work rate increased by 0.5 W/second until cycling cadence fell below 60rpm. During all exercise tests, athletes wore a face mask (7450 V2 Mask; Hans Rudolph, Inc., Shawnee, KS), which was connected to a metabolic cart (TrueOne 2400; ParvoMedics, Sandy, UT, USA).   4.2.7 Time trial visit All athletes performed a high-intensity, short-duration exercise bout in the form of a simulated 10-km time trial on a cycle ergometer. Upon arrival in the laboratory, baseline spirometry was assessed in triplicate. During the time trial, a virtual course was displayed on a screen, with distance, cadence, and gearing information displayed. Immediately following the time trials, spirometry was repeated. On average, men and women completed the 10-km time trials in        15 minutes 39 seconds and 17 minutes 54 seconds, respectively. Respiratory parameters (RR, Vt, and VETT) are reported as means from the final two kilometers of the time trial. Male and female athletes completed the final two kilometers of the 10-km time trial in 3 minutes 6 seconds and    3 minutes 30 seconds, respectively.  66  4.2.8 Comparisons of targeted and achieved bronchoconstriction stimulus by EVH challenge The VEEVH-target was normalized by VEGXT to generate an index of the relative intensity of VEEVH-target. Similarly, the intensity of the bronchoconstriction stimulus of VEEVH-achieved was evaluated by normalizing VEEVH-achieved to several reference parameters:  (1) to VEGXT and VEEVH-target by calculating the percent VEEVH-achieved of VEGXT and of     VEEVH-target, respectively;  (2) to FEV1 baseline and expressed as a product thereof;  (3) and stated as percent of predicted maximum voluntary ventilation (MVV), where MVV predicted was calculated as 35 x FEV1 at baseline.  4.2.9 Statistical analyses Anthropometric, spirometric, and baseline fitness parameters were compared between male and female athletes using the independent-samples t-test. To compare the FI calculation methods with each other and to the change in FEV1 following the time trial, we used repeated-measures ANCOVAs with sex as the covariate. To assess the mean variation between the four FI calculation methods, we determined the difference between the highest and the lowest FI for every individual, and calculated the average of these differences across all athletes. The Friedman test was used to compare the number of EVH+ and EVH- tests between the four FI calculation methods, and to compare a cutoff of FI ≥ 10% to a cutoff of FI ≥ 15%. Before running the Friedman test, all assumptions were confirmed to be met. If a significant main effect was found, post-hoc analysis was performed using the Wilcoxon signed-rank test with a 67  Bonferroni correction applied. We followed a conservative approach when interpreting EVH tests as EVH+ vs EVH-. The FI values were assessed on the basis of one decimal, which were not rounded. For example, using a cutoff of 10% an FI of 9.8% was considered a negative test.  We used repeated-measures ANOVAs to compare the VEs generated by the EVH challenge, the GXT, and time trial with one another. The Shapiro-Wilks W test for normality and Levene test for equal variances were used to confirm that the assumptions of ANCOVA were met. If a main effect was found, a Tukey’s HSD test was performed to test for significance. Linear regression was used to compare predicted MVV to VEGXT and to compare the effect of VEEVH-achieved on the FI calculation methods. For all tests, the significance level was set at p < 0.05. Data are presented as means and standard deviations. Statistical procedures were completed using SPSS (V24.0; IBM, Armonk, New York).  4.3 Results 4.3.1 Study participant characteristics Of the included 37 athletes, five had a previous history of asthma. Three of the five athletes with a previous asthma diagnosis treated their symptoms at the time of study participation with daily ICS and short-acting IBAs on an as-needed basis. Another two athletes relied on short-acting IBAs on an as-needed basis only. All anthropometric and fitness parameters collected are summarized in Table 6. Men were significantly older (p = 0.037), taller (p < 0.001), and heavier than women (p < 0.001). As a result, VO2max (p < 0.001), absolute (p < 0.001), and relative power (p = 0.004) were significantly greater in male compared to female athletes. There were no statistical differences in weekly training volume and years of training between male and female athletes (see Table 6).  68  Table 6: Anthropometric, pulmonary, and fitness characteristics of 18 male and 19 female athletes. Variable M (SD) Min - Max Age (years)a Male Female 29 (6) 31 (6) 27 (5) 19 – 45 22 – 45 19 – 37   Baseline FEV1 (% predicted) Male Female 111 (13) 109 (9) 113 (15) 89 – 154 89 – 125 91 – 154  Baseline FEV/FVC (% predicted) Male Female 94 (8) 94 (9) 94 (7) 89 – 119 77 – 119 80 – 103   Height (cm)b Male Female 173 (9) 179 (5) 167 (6) 157 – 188 170 – 188 157 – 175  Mass (kg)c Male Female 67 (9) 74 (6) 61 (5) 54 – 90 59 – 90 54 – 68  Training load (hours/week) Male Female 12 (5) 11 (5) 13 (4) 6 – 23 6 – 23 7 – 20  Years of consistent training Male Female 5 (4) 5 (3) 5 (4) 1 – 20 2 – 14 1 – 20  VO2max (mL/kg/min)d Male Female 59.1 (7.0) 64.6 /5.2) 54.0 (3.9) 48.9 – 77.7 56.6 – 77.7 48.9 – 63.1  Maximal power (W)e Male Female 375 (58) 428 (32) 324 (13) 297 – 502 373 – 502 297 – 346  Relative power (W/kg)f Male Female 5.6 (0.5) 5.8 (0.5) 5.4 (0.4) 4.6 – 6.5 4.8 – 6.5 4.6 – 6.1   FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity; VO2max, maximal oxygen consumption.   a Men were significantly older than women (p = 0.037). b Men were significantly heavier than women (p < 0.001). c Men were significantly taller than women (p < 0.001). d Men’s VO2max was significantly increased compared to women’s VO2max (p < 0.001). e Maximal power was significantly greater in male compared to female athletes (p < 0.001). f Relative power was significantly greater in male athletes compared to female athletes (p = 0.004). 69  4.3.2 Comparisons of EVH FI calculation methods and time trial spirometry There was a significant main effect for FI between the four calculation methods (p = 0.020). Compared to FIA and FIB using FID led to significantly greater FIs (p = 0.017 and p = 0.032, respectively). The FIC was significantly greater compared to FIA (p = 0.044), but not to FIB            (p = 0.083). Furthermore, there were no statistical differences between FIA and FIB (p = 0.324), between FIC and FID (p = 0.183). On average, the difference between the highest and lowest FI calculated for every participant with FIA-D was 6.0 (7.3)% (see Figure 8 and Table S1 in the Appendix). No correlations were found between VEEVH-achieved and the FI, regardless of the applied FI method (FIA: r =0.105, n = 37, p = 0.536; FIB: r =0.138, n = 37, p = 0.416;               FIC: r =-0.069, n = 37, p = 0.684; FID: r =-0.116, n = 37, p = 0.494).   Analyzing the EVH challenge with FIA, FIB, FIC, and FID led to EVH+ results suggestive of EIB+ in 9, 10, 13, and 16 athletes, respectively (see Table 7). The ratios of EVH+ and EVH- tests between the four FI calculation methods did not differ statistically from one another. As shown in Figure 7, increasing the EVH+ cutoff from 10% to 15% for FIA, FIB, FIC, and FID led to a significant reduction of the positives to 2, 3, 3, and 10, respectively (χ2 (37) = 53.9 p < 0.001). Post-hoc analysis with the Wilcoxon signed-rank test was conducted with a Bonferroni correction applied, resulting in a significance level set at p < 0.013. When comparing a cutoff of FI ≥ 10% to a cutoff of FI ≥ 15%, significant differences in the number of positives and negatives were found for FIA (Z = -2.65, p = 0.008), FIB (Z = -2.65, p = 0.008), and FIC           (Z = -3.16, p = 0.002), but not for FID (Z = -2.449, p = 0.014).   70  Eight athletes tested EVH+ in all four FI calculation methods. The VEEVH-achieved did not differ between these eight EVH+ athletes and the 29 EVH- athletes. Two athletes with no prior diagnosis of EIB tested EVH+ in all four calculation methods with an FI ≥ 15%. However, these two athletes experienced mild exercise-induced bronchodilation immediately following the time trial (increase in FEV1 of 4.31 and 1.95%, respectively). Six of the eight previously mentioned EVH+ athletes presented with a FI ≥ 10% but < 15%, which resulted in an EVH+ diagnosis using FIA-D. Among those six athletes were four with a previous history of EIB+. Two of the four athletes with a previous EIB+ diagnosis experienced mild EIB following the completion of the time trial (decrease in FEV1 of -1.7% and -2.2%), while the other two athletes underwent bronchodilation of 8.6% and 2.5%. Three athletes tested EVH+ following the calculations of FIC and FID only, all experiencing bronchodilation following the time trial (increase in FEV1 of 2.6, 8.58, and 2.17%).    Figure 7: Number of EVH+ and EVH- tests based on a cutoff of 10% and 15% using the FIA, FIB, FIC, and FID calculation method, respectively.  * There was a significant difference in the number of EVH+ and EVH- tests when comparing a cutoff of FI ≥ 10% to a cutoff of FI ≥ 15% in the interpretation of the FIs using FIA, FIB, and FIC, respectively. 71  In total, there were seven athletes presenting with bronchoconstriction (mean decrease in FEV1 of 4.4 (3.2)%) after the completion of the time trial with a decrease in FEV1 ranging between 1.2% and 9.1%. The remaining 30 athletes bronchodilated with an increase in FEV1 of 5.7% (6.7%), ranging from 1.1% - 31.9%.  Figure 8: Fall indices calculated using four different methods for every athlete.    72  Table 7: Respiratory parameters for EVH challenge, maximal graded exercise test, and time trial. Respiratory Challenge Vt (L) M (SD) RR (b/min) M (SD) VE  (L/min) M (SD) VE/baseline FEV1  M (SD) VEEVH-target  Total Men Women      131 (24) 146 (19) 116 (19)  30 30 30 VEEVH-achieved Total Men Women  2.2 (0.6) 2.6 (0.5) 1.8 (0.3)  55 (8) 54 (10) 56 (67)  98.0 (20) 114 (14) 83 (10)  23 (3) 24 (2) 22(3) VEGXT Total Men Women  2.8 (0.6) 3.2 (0.5) 2.5 (0.4)  65 (18) 68 (24) 63 (10)  151 (29) 173 (25) 131 (16)  35 (5) 36 (3) 35 (6) VETT Total Men Women  2.5 (0.6) 2.9 (0.5) 2.0 (0.3)  50 (11) 48 (12) 53 (10)  120 (28) 135 (27) 105 (20)  28 (7) 28 (5) 28 (6)  EVH, eucapnic voluntary hyperpnea challenge; VEEVH-target, target minute ventilation during the EVH challenge; VEEVH-achieved, average minute ventilation achieved during the eucapnic voluntary hyperpnea test, VEGXT, highest 15-second average for minute ventilation achieved in the final minute of the graded maximal exercise test; VETT, average minute ventilation achieved during the final two kilometers of the 10-km time trial, Vt, tidal volume; RR, respiratory rate; VE, minute ventilation; FEV1, forced expiratory volume in 1 second.  4.3.3 Comparison of minute ventilations between EVH challenge and maximal graded exercise test, a short-duration, high-intensity cycle bout, and predicted MVV The VEs generated by the EVH challenge, the GXT and time trial are presented in Table 7. The VEGXT was 101 (13)% of predicted MVV, and both parameters (VEGXT and predicted MVV) were positively correlated (r = 0.751, n = 37, p < 0.001). The VEEVH-target equalled 87 (13)% of VEGXT: 89 (17)% in female and 84 (8%) in male athletes (see Figure 9). Two athletes fell outside of the 95% confidence interval. 73  Despite feedback and coaching, none of the athletes maintained VEEVH-target over the final five minutes of the EVH challenge. On average, athletes sustained 75 (9)% of their VEEVH-target. As a function of body surface area and lung size, VEEVH-achieved was significantly higher in male (112.3 (14.3) L/min) than in female athletes (83.7 (10.0) L/min; p < 0.001); however, there was no sex difference when VEEVH-achieved was normalized by VEEVH-target between male (77 (9%)) and female athletes (73 (10)%, p = 0.188). When expressed as a product of FEV1, women’s      VEEVH-achieved corresponded to 22 (3) x FEV1, which was significantly lower than the 24 (2) x FEV1 maintained by men (p = 0.041). Identically, normalizing VEEVH-achieved by predicted MVV, women achieved 62 (8)%, which was significantly lower compared to 68% (7%) in men            (p = 0.014). Relative to VEGXT, women’s percent VEEVH-achieved (64 (9)%)  and percent VETT (87 (21)%) were higher compared to men’s VEEVH-achieved (66 (9)%) and VETT (75 (19)%), but these values did not reach statistical significance (p = 0.389 and p = 0.064, respectively). All athletes were eucapnic during the EVH challenge with a mean fraction of exhaled CO2 of 5.2 (0.9)%.   Figure 9: Target ventilation of EVH challenge (VEEVH-target) normalized by ventilation achieved on the graded maximal exercise test (VEGXT).  A: Histogram of normalized VEEVH-target. B. Dispersion of VEEVH-target after normalizing data to individual VEGXT. The center vertical line represents the median; the boxed region indicated the interquartile range, lines extending from the box indicate the 5th - 95th percentile range; dots indicate values lying outside the 5th – 95th percentile range.  74  4.4 Discussion The findings of this study are: (1) There are significant differences between the four FI-calculation methods, with an intra-individual range of 6.0 (7.3)%, affecting the number EVH+ challenges (9, 10, 13, and 16 EVH+ tests using FIA, FIB, FIC, and FID, respectively). (2) There was no correlation between VEEVH-achieved and FI, regardless of the calculation method used. (3) Despite being trained athletes, none of the participants in this study maintained VEEVH-target in the EVH challenge. The VEEVH-target equalled 87 (13)% of VEGXT and 114 (36)% of VETT, highlighting the relatively aggressive bronchoconstriction stimulus placed on the airways by the EVH challenge. (4) When normalized by VEGXT, the relative intensity of the bronchoconstriction stimulus with VEEVH-target calculated as 30 x FEV1 leads to a considerable interindividual range (67% - 135%). This confirms previous studies113 and suggests that calculating VEEVH-target as a product of FEV1 could decrease the diagnostic accuracy of the EVH challenge. (5) A short-duration, high-intensity cycle bout led to exercise-induced bronchodilation in 30/37 (81%) athletes, regardless of their FI in the EVH challenge. No athlete decreased FEV1 by 10% or more following the time trial.  4.4.1 Comparison of FI-calculation methods Our study shows that the calculation method applied to determine the FI can lead to a variation in the FI by up to 6%, which will impact the interpretation of the EVH challenge, and as a result the calculation needs careful consideration. Basing FI on two consecutive FEV1 values post-hyperpnea with a decrease ≥ 10% either between two time points (FIA) or within one time point (FIB) by assessing the FEV1 measures that are taken in duplicates individually, results in comparable FIs (mean difference between FIA and FIB = 1.4 (1.7)% and EVH+ diagnoses         75  (FIA = 9 EVH+; FIB = 10 EVH+)). Four athletes tested EVH+ using FIC but tested EVH- using FIA. The average difference between FIA and FIC for those four athletes is 9.8%, allocating them into the previously mentioned intermediate group of EIB+ athletes with an FI between 10 and 15%. With only one post-hyperpnea FEV1 value ≥ 10% and bronchodilation following the TT in all but one (M10, see Supplemental Table S1 in the Appendix) athletes, FIC appears to be affected by low FEV1 values that were the result of poor technique.  Adjusting FI for VEEVH-achieved (FID) led to an average difference of 21% between FID and FIC. This finding is in contrast to Hurwitz et al.42 who compared raw FIs (equivalent to FIC in this analysis) with FIs normalized for VEEVH-achieved in a study population that included older participants and individuals with a smoking history. They only found a 1% difference between their raw and adjusted FIs.42 The reasons for this disparity are unclear; however, the current study examined young, athletic non-smokers. As such, the percent predicted FEV1 of athletes in this study was considerably greater compared to that of Hurwitz’ study population (FEV1 percent predicted in healthy individuals and asthmatics = 98 (2)% and 91 (2)%, respectively).42 With such high FEV1 values in the athletes, the EVH targets are much larger than in the Hurwitz population, which could explain the low relative VEEVH-achieved in this study, and subsequently, the greater difference in FID. Adjusting FI for VEEVH-achieved in trained, healthy athletes with high percent predicted values for FEV1 seems to give quite different results compared to the raw FI values, and thus, such a method should be used with caution.   76  4.4.2 Bronchoconstriction stimulus induced by EVH challenge, GXT, and TT   Evaporative water loss from the airway surface is thought to be the main stimulus for EIB.35 Both the exercise and EVH challenges are designed to stimulate airway surface water loss with subsequent bronchoconstriction through high VEs. Argyros et al.44 tested various EVH dosing schedules to evaluate the duration and absolute VEs as correlates of bronchoconstriction. A hyperpnea challenge of six minutes at a VEEVH-target of 30 x FEV1 led to significantly greater levels of bronchoconstriction compared to a six-minute challenge at 20 x FEV1.44 Despite real-time feedback, coaching, and verbal encouragement in this study, none of the athletes were able to maintain 30 x FEV1 in the final five minutes of the EVH challenge. The range of VEEVH-achieved relative to VEEVH-target was 58% - 91%, corresponding to an average of 23 x FEV1. Surprisingly, we did not find a correlation between VEEVH-achieved and the FI, regardless of the FI-calculation method. One reason for the absence of a correlation between VEEVH-achieved and the calculated FIs could be that 30 x FEV1 is significantly greater than VETT and 87% of VEGXT; therefore, a bronchoconstriction stimulus induced by 75% of VEEVH-target might still be sufficiently powerful to screen for EIB. Healthy individuals and elite athletes can achieve between 60% and 90% of their predicted MVV in an exercise bout.46 Anderson and Kippelen35 state that for non-habitual exercisers, a target ventilation of 17-21 x baseline FEV1, or 60% of predicted MVV, is a sufficiently potent stimulus for an EVH test. Despite the athletes’ fitness levels, weekly training volume and years of training history as endurance athletes, the participants of this study only averaged a VE of 65% of predicted MVV, indicative of a sufficiently high stimulus to induce bronchoconstriction, but ranging in the low-end of the desired range for this group.   77  Spiering et al.113 investigated the efficacy of 30 x FEV1 as a standardized VEEVH-target in Olympic-level endurance athletes. Similarly to Spiering et al.113 we found a significant positive correlation between VEGXT and predicted MVV and a wide range (VEEVH-target relative to VEGXT; min -      max = 67% - 135%) in the bronchoconstriction stimulus that trained athletes were supposed to generate in the EVH challenge. In the Spiering study, the VEEVH-target in approximately 40% of the Olympic-level athletes was inappropriately low, risking false negatives.113 Spiering et al.113 recommend the use of 85% of VEGXT for athletes for whom VEGXT is available. In our study, one athlete was below the 5% confidence interval and thus, at risk for an inadequately low bronchoconstriction stimulus during the EVH challenge. The VEEVH-target of another athlete was exceeding the 95% confidence interval, and therefore not representative of a naturally occurring bronchoconstriction stimulus. Our study supports the recommendation of using a percentage of VEGXT as a more individualized VEEVH-target, particularly since 85% of VEGXT equals 96 (25)% of VETT, making it an achievable hyperpnea target.  Based on the results from this study, it is unclear if there are sex differences in the bronchoconstriction stimulus that was generated during the EVH challenge. Women’s        VEEVH-achieved, expressed as a product of FEV1 and as a percent of predicted MVV, was significantly smaller than that in men of this study, which is identical to the findings by Brummel et al.114 However, when expressing VEEVH-achieved as a percentage of VEGXT or VEEVH-target, there were no sex differences. When mimicking submaximal and maximal exercise, the total amount of O2 required by the respiratory muscles and the fraction of whole-body VO2 dedicated to meet the metabolic demands of the respiratory muscles (VO2RM) are significantly greater in women compared to men.115 Particularly at VE > 60 L/min, VO2RM is disproportionally greater in 78  women compared to men, which was exceeded by all athletes in this study for VEEVH-achieved. Smaller lung volumes when matched for height and smaller airways when matched for lung size are the anatomical causes for the lower ventilatory capacity and the mechanical respiratory disadvantage in women compared to men.64,69 Despite a lower relative VEEVH-achieved expressed as a product of FEV1, or relative to predicted MVV, it could be possible that the mechanical and metabolic demands placed by the EVH challenge on the respiratory system are comparable, or possibly even substantially higher, in women compared to men. Further research investigating possible sex differences in the ventilatory mechanics during the EVH challenge is required to answer the questions of (1) whether increased respiratory mechanical strain results in an increased airway surface liquid loss, leading to a greater bronchoconstriction stimulus in women than in men at comparable target VEs; and (2) whether with smaller airways in women compared to men, smaller changes in diameter, i.e. smaller degrees of airway smooth muscle contractions, are needed to induce similar reductions in FEV1, thus affecting the EVH outcome.   4.4.3 Bronchodilatory vs. bronchoconstrictive stimulus of reflex-induced hyperpnea vs. mimicked hyperpnea Following the completion of the time trial, none of the athletes included in this study showed a reduction in FEV1 ≥ 10%. This finding deserves further exploration. Possible explanations could relate to either the relative mildness of the EIB in this population, or perhaps the inadequacy of a time trial as an asthmogenic stimulus in fully warmed-up athletes. Asthma is a heterogeneous condition with variable airway diameter and function as a common feature.116 Similarly, airflow obstruction has been shown to be variable in athletes with EIB, particularly with mild EIB.23,33 Mild EIB may be a different phenotype from moderate or severe EIB.33 For example, researchers 79  and physicians are divided on whether athletes with mild EIB (with or without symptoms) should be treated with IBAs.51 Ultimately, this question is linked to the discussion of whether a decrease in FEV1 ≥ 15% on the EVH test is a physiological response in non-symptomatic individuals,47,105 particularly when considering the poor reproducibility of the EVH challenge in recreational athletes when a cutoff value of 10% in FEV1 is employed.104  Given that the spirometric measures performed pre- and post-time trials with a prevalence of bronchodilation of 81% did not backup the number of positives in the EVH challenge, regardless of the applied cutoff, we are hesitant to make a statement on the proposed increase in the EVH cutoff. Upon first sight, it may look like a shift from a 10% to a 15% cutoff would reduce the rate of false-positives based on decreased number of 9, 10, 13, and 16 EVH+ tests to 2, 3, 3, and 10 EVH+ tests using FIA, FIB, FIC, and FID, respectively. However, the exercise-induced bronchodilation following the time trials was independent of the FI calculations. Based on our data, we do not feel comfortable answering the questions of whether a decrease in FI of 10-15% is a physiological response to EVH, and whether an increase of the EVH+ cutoff  to 15% serves to decrease the number of false-positive tests. Further research comparing the bronchoconstriction stimulus induced by the EVH challenge and exercise protocols are needed.  The purpose of the time trial was to generate cardiorespiratory data in a sport-specific, high-intensity, short duration exercise bout, not to induce EIB. Contrary to exercise challenges with the intention to screen for EIB, all athletes participating in this study were well warmed-up prior to starting the time trial, minimizing the presentation of EIB. A thorough warm-up prior to high-intensity exercise has been shown to reduce the risk of EIB in athletes with AHR.49  80  This study underlines the potent bronchodilatory stimulus of high-intensity, short-duration exercise. In the present study, two out of seven athletes with a mild decrease in FEV1           (mean = -4.4%, ranging from -1.2% to -9.1%) had a previous diagnosis of asthma. All remaining 30 athletes, including six of the eight athletes with an EVH+ diagnosis on all four FI calculation methods, presented with exercise-induced bronchodilation. This is in contrast to the findings by Barnes et al,117 who reported a similar bronchoconstriction response in the voluntary hyperventilation and the exercise stimulus in asthmatic subjects. Comparisons between Barnes’ et al.117 study participants and the athletes of this study are difficult because parameters relevant in the description of EIB+ individuals such as exercise history, training status, and warm-up prior to the treadmill test are not provided for the 16 year-old asthmatic and non-asthmatic study participants. Voluntary hyperpnea differs from reflex-driven, whole-body exercise hyperpnea. In the Barnes et al. study,117 adrenaline and noradrenaline levels were significantly increased following the treadmill test but not following mimicked hyperpnea. These findings underline a greater sympathetic response to a maximal exercise effort, resulting in higher adrenaline and noradrenaline levels, thus causing greater levels of bronchodilation compared to voluntary hyperpnea. There are further differences between voluntary hyperpnea and reflex-driven hyperpnea from a respiratory mechanics point of view.118,119 Dominelli et al.118 showed that even when VE, RR, and Vt during voluntary hyperpnea were in close agreement with the values generated during submaximal and maximal exercise, EELV, WOB, and VO2RM were higher during voluntary hyperpnea due to excessive pressure generated on expiration. When practicing how to mimic submaximal exercise ventilations in the study of Dominelli et al.,118 subjects inhaled the target volume of air faster than in the exercise trial, resulting in higher initial airflows and subsequent pre-expiratory pauses. As previously mentioned, it is unclear if these differences 81  in respiratory mechanics and breathing patterns in voluntary hyperpnea and maximal exercise affect the amount of evaporative airway surface water loss by possibly changing the characteristics of airflow (increase in turbulent vs laminar flow) and the amount of dead-space ventilation (increase in airway cooling) and thus, the bronchoconstriction stimulus induced.  4.4.4 Limitations Fourteen athletes inhaled 200 µg of salbutamol following the EVH challenge prior to the GXT. The purpose of the GXT was to assure that athletes’ fitness levels met the original studies’92,111 inclusion criteria. To keep the time commitment of the study participants within a manageable range, we decided to conduct the GXT following the EVH once FEV1 was within 150 mL or 5% of baseline. Due to the bronchodilatory effect of this SABA, VEGXT may have been greater for those athletes than the VEs that would have been achieved without prior medication intake; however, there was no statistical difference in VEGXT of these 14 athletes compared to the remaining 25 athletes.  Another limitation of this analysis is the variation in the recovery time between the completion of the EVH challenge and the start of the GXT. Instead of following a predetermined timeline, we decided to continue the GXT when athletes’ FEV1 was within 150 mL or 5% of baseline or when athletes reported that they felt sufficiently recovered (provided their FEV1 value had returned to baseline levels). The male athletes included in this analysis performed a practice time trial following the GXT on the screening day, whereas female athletes did not. In this retrospective analysis, we analyzed the VEs generated in the final two kilometers of the 10-km time trials as a sample of the VEs athletes sustain in a short duration, high intensity exercise bout. While there may be a benefit of 82  adding a practice time trial to the assessment day in an effort to reduce a learning effect on pacing strategy and mean power output on the time trial day, there is likely little effect of a practice time trial on the VEs in the final two kilometers in this population, as trained athletes with experience in racing time trials will be adept at pacing for the required durations, as indicated by the RPEL values in Chapters 2 and 3.  We would like to acknowledge that male athletes followed a 20-minute predetermined warm-up protocol outlined in section 3.2.4, whereas female athletes completed a 15- to 20-minute self-directed warm-up. All athletes were experienced cyclists or triathletes who were competing at the time of study participation. No record was kept of the exact warm-up that the female athletes completed; however, based on observations, female athletes completed a standard warm-up which included short bouts of high intensity, making their warm-up similar to the predetermined protocol of the male athletes.  Lastly, we collected spirometric data up to 20 minutes following the EVH challenge but performed just a single spirometry assessment with two measures immediately following the time trial. Blackie et al120 concluded that the onset of bronchoconstriction was delayed with increasing duration of isocapnic hyperventilation in asthmatics. Hyperventilation itself may inhibit bronchoconstriction, or the mechanisms inducing bronchoconstriction may be delayed in response to hyperventilation.120 It would have been preferable to have spirometric data following the time trial over a comparable 20-minute time period as for the EVH test; however, eight out of nine EVH+ athletes (according to FIA) presented with their greatest fall in FEV1 within the first five minutes following EVH. Similarly, these findings could suggest that the greatest fall in FEV1 following the time trial can be expected within the first five minutes upon exercise cessation.  83  4.5 Conclusions The endurance athletes of this study did not achieve VEEVH-target. It is possible that the target calculated as 30 x FEV1 overestimates achievable VEs for voluntary hyperpnea, particularly in individuals with percent predicted values for FEV1 ≥ 100% predicted. The FI calculations were not affected by VEEVH-achieved, provided that 60% of predicted MVV or 17 x FEV1 at baseline was maintained. Attention is needed when choosing the calculation method of the FI. Among the four calculation methods compared in this study, FI varied by an average of 6% between the four calculations methods, resulting in 9, 10, 13, and 16 EVH+ tests using FIA, FIB, FIC, and FID, respectively. Adjusting FI for the intensity of the bronchoconstriction stimulus by normalizing FI to VEEVH-achieved may lead to significantly higher FI values, possibly risking false-positive EVH+ results. Assessing FI on the basis of two consecutive FEV1 values with a decrease ≥ 10% either between (FIA) or within two time points (FIB) appears to be superior to FIC and FID. Further research is required to better understand the phenotype of mildly EIB+ athletes and their airway responses to EVH and various exercise protocols. 84  Chapter 5: Conclusions  5.1 Overall conclusions Athletes using IBAs appear to win a disproportionally greater percentage of individual medals at Olympic Games compared to athletes who do not use IBAs.15,121 In the past, research on the ergogenic effects of IBAs on athletic performance has focused on male athletes.60-62 Due to anatomical and functional differences in the respiratory response to exercise, women have the potential to receive a greater benefit from IBAs compared to men. As outlined in Chapter 2, we found a significant increase in FEV1 after the inhalation of 400 µg of salbutamol in trained female athletes, which was independent of EVH status. Despite this increase in lung function, mean power output during a 10-km time trial was decreased. When comparing these findings from female athletes to the results of a previous study, which used an identical study design with trained male athletes,60 there were no differences in the bronchodilatory effect of 400 µg of salbutamol on FEV1. In men, the inhalation of salbutamol did not affect time trial performance, or other parameters relevant for athletic performance such as VO2.60 In women, a significant increase in VO2, and a decrease in cycling economy, and symptoms such as restlessness and tremor suggest an overstimulation of women’s adrenergic β2-system due to an increased relative salbutamol dosage compared to men (relative to BM).  These findings lead to the rejection of the first and second hypotheses of Chapter 2. Specifically, we observed a significant increase in lung function, assessed by FEV1, in female and male athletes after the inhalation of 400 µg salbutamol; however, this increase in FEV1 did not translate to an enhanced mean power output in the 10-km time trial performance. On the 85  contrary, in female athletes, we observed a decrease in mean power output and cycling economy following IBA use.   Contrary to the two hypotheses of Chapter 3, the inhalation of the maximally allowed daily dose of salbutamol of 1600 μg did not improve 10-km time trial performance in trained male athletes, regardless of EVH status. Furthermore, athletes with a smaller BM in Chapter 3 did not show an ergogenic benefit due to the greater relative dose of IBAs they received compared to athletes with a greater BM. This finding supports WADA’s approach of setting an upper limit to the permitted daily dose of salbutamol as an absolute dose instead of relative to BM. It was beyond the scope of this thesis to investigate how the salbutamol dose relative to BM affects urine levels, which are used to assess athletes’ adherence to WADA’s upper limit, a method that has been questioned.122-124 The threshold of 1000 ng of salbutamol per milliliter of urine has been exceeded even when adhering to the maximally allowed daily dose.122,123 Dehydration and physical activity have been shown to affect salbutamol levels in urine.122,123 As outlined in section 1.1.3, athletes have the right to prove that abnormally high urine levels were the result of an inhaled therapheutic dose; however, this is costly and adds psychological stress to athletes. Setting an upper limit of the maximal daily dose of IBAs based on BM would lead to further complications due to intra-individual fluctuations in BM and due to subsequent adjustments to individualized urine cutoffs with respect to AAF-testing.  Significant increases in HR, VE, RR, and RPEL after the inhalation of 1600 μg of salbutamol in the male athletes included in Chapter 3 appear to be indicative of an overstimulation of the adrenergic nervous system, as with the female athletes in Chapter 2 using a quarter of the salbutamol dose. There does not appear to be a linear relationship between IBA dose and induced 86  bronchodilation when comparing the increases in FEV1 following 1600 μg of salbutamol to a previous study from our laboratory, in which we assessed lung function and athletic performance after the inhalation of 400 μg.60  We accept the two hypotheses of Chapter 4 in which we retrospectively compared four previously reported methods of determining the lowest FEV1 for the calculation of the FI in the EVH challenge. There were significant differences between the FIs when calculated following four different methods, suggesting that attention is needed when determining the FI method and comparing FIs between studies. Among the four calculation methods compared in this thesis, there was an average difference of 6% between the highest and the lowest FI calculated for every athlete affecting the EVH interpretations. Using the FIA, FIB, FIC, and FID methods with a cutoff of 10% led to 9, 10, 13, and 16 positives, respectively. It appears that particularly when athletes have high percent predicted values for FEV1, adjusting FI by VEEVH-achieved may lead to significantly higher FI values, possibly risking false-positive EVH+ results. High percent predicted values in FEV1 in the athletes of this thesis were likely also the reason for why individuals did not achieve VEEVH-target during the EVH challenge. On average, the VEEVH-target exceeded the VETT by 11 L/min, emphasizing the powerful bronchoconstriction stimulus that is placed on an athlete in the EVH challenge. Our findings also highlight the powerful bronchodilation stimulus of exercise, as shown by the high prevalence of 30/37 athletes (81%) who demonstrated an increase in FEV1 following the time trial, independent of any FI calculation method.   87  5.2 Future directions 5.2.1 Future research on the ergogenic potential of IBAs In the absence of evidence for an ergogenic potential, the data presented in this thesis do not support the hypothesis that inhaled salbutamol enhances athletic performance in exercise bouts of high intensity, lasting approximately 15 minutes, even when using the maximally allowed daily dose following the WADA guidelines. The studies on the ergogenic potential of salbutamol in EIB+ and EIB- female (Chapter 2) and male (Chapter 3) athletes highlight the importance of a warm-up. The warm-up prior to the time trials following the inhalation of the placebo appeared to be of a bronchoprotective nature in EIB+ and EIB- athletes. The warm-up is an often neglected but important strategy in the management of EIB.49 All athletes included in the studies of this thesis performed either a self-directed (female athletes in Chapter 2) or a predetermined (male athletes in Chapter 3) 15 - 20 minute long warm-up prior to the time trials. In the presence of this warm-up, there appears to be no benefit of salbutamol to athletes with EIB in terms of symptoms or performance. If the subjects had not warmed-up, the potential for a greater effect of salbutamol might exist. Further research to investigate various warm-up exercise protocols, particularly in athletes with mild EIB, is needed. In their review on the effect of the warm-up on EIB, Stickland et al.49 drew attention to the low sample size of most of the included studies. Therefore, a focus on sufficiently powered studies will be important to gain further insight on the effects of various warm-up protocols on EIB. Furthermore, studies investigating the mechanisms of a potential bronchoprotective effect of a warm-up in EIB+ athletes are needed. A better understanding of the bronchoprotective mechanism of a warm-up in EIB+ athletes prior to training or competition could reduce the amount of IBAs needed to treat EIB symptoms and thus, minimize the risk of the known ß2-receptor downregulation.52  88  The inhalation of 400 μg and 1600 μg of salbutamol prior to exercise by female and male athletes, respectively, did not lead to the hypothesized performance enhancements; rather, they led to a high incidence of side effects such as tremor, restlessness, and increased resting HR in female and male athletes. Future studies on the ergogenic effects of IBAs, particularly high dose IBAs, should take the systematic assessment of these potentially deleterious factors into consideration to provide athletes, physicians, and coaches with further insight when managing athletes with EIB.  With a focus on athletic performance as the main outcome, respiratory mechanics were not evaluated in this thesis. Studies assessing EFL, WOB, and VO2RM during sport specific exercise bouts in trained athletes following IBA use are needed to evaluate their effects on respiratory mechanics and to determine if there are sex differences in the response to IBAs from a breathing mechanics point of view. Additionally, the focus on athletic performance enhancement in this thesis was placed on 10-km cycling time trials (average completion time for male and female athletes > 15 minutes) thirty minutes following IBA use. Salbutamol, like other SABAs, is used as a ‘rescue’ medication to induce immediate bronchodilation during acute EIB. Further research is needed to determine the ergogenic effect of high doses of IBAs on shorter (< 2 minutes) duration and intermittent intensity exercise protocols immediately following the inhalation.   To conclude this section on future research that is needed to gain a better understanding of the ergogenic effects of IBAs, we would like to draw attention to statistical and practical challenges scientists face when assessing the enhancement of sport performance. Similar to many interventions in medical research, scientists interested in the ergogenic effect of pharmacological 89  agents have to ask themselves whether statistical significance equals practical relevance. In many cases this question is answered with no. Wasserstein and Lazar125 define the p-value as “the probability under a specified statistical model that a statistical summary of the data (e.g., the sample mean difference between two compared groups) would be equal to or more extreme than its observed value.” As such, p-values do not measure the size of an effect or the importance of a result. In the studies presented in this thesis, athletic performance was decreased by 1.1% in female athletes (Chapter 2), and increased by 0.5% in male athletes (Chapter 3). In the past three Summer Olympic Games, the average difference between the gold and silver winning performance in the road cycling time trial was 1.1% and 0.7% in male and female athletes, respectively (see Table 8). Due to variability in the measurement technique and equipment, in addition to within-athlete variability in a given sport performance (for example a 10-km time trial), and small sample sizes, such small differences might not lead to a statistically significant finding with a p < 0.05 in a typical research study.126 To determine “worthwhile enhancements of performance” for athletes, one needs to know the within-athlete standard deviation for a given performance and the between-athlete variation (i.e. the variation in ability between athletes) to define the anticipated spread between athletes at the finish of an event.126 The greater the difference in athletic performance between athletes relative to the within-athlete repeatability, the greater the ergogenic effect needed to help an athlete win first place from a lower ranking. To apply this advice to future studies on the ergogenic effect of IBAs, individualized “worthwhile enhancement” thresholds could be calculated and used as a reference when determining the ergogenic potential, in addition to traditional p-values. It would be interesting to record within-athlete and between-athlete variation for a race that is repeated in regular (ideally weekly or bi-weekly) intervals using the identical course to determine individualized thresholds. However, a 90  challenge when recruiting high-level athletes for research projects, particularly when using maximal daily doses of pharmaceutical agents that are on the list of prohibited substances by WADA,39 is fear of AAFs in case of doping controls. Furthermore, many athletes competing at high levels adhere to routines which may not be compatible with study protocols, making athletes hesitant to participate in studies during their racing season. Large variations in fitness levels based on the periodization of their training plan reduces the validity of studies investigating the ergogenic potential of pharmaceutical agents in athletes’ off-season.126  In both our studies, athletes were racing against themselves with minimal feedback (distance, cadence, and gearing information) on the screen from the 3D cycling software. Laboratory-based time trials differ from actual time trial races in the field, where athletes know their competitors’, and their own split times, and their real-time power outputs. The presence of spectators and the fact that athletes are racing for prize money or qualification points add to the racing atmosphere. We agree with Hopkins et al.126 and Kearney,127 and suggest that future research on the ergogenic potential of IBAs should be complemented by studies in the field in actual race settings. Due to a decreased within-athlete variability, field settings may be superior to a simulated race in the laboratory.  91  Table 8: Finishing times and percent differences between the gold, silver, and bronze performances in the road cycling time trials of the past three Olympic Games for male and female athletes.  Sex and Ranking Rio 2016 Finishing Time (h:min:s:ms) Differences   (%) London 2012 Finishing Time (h:min:s:ms) Differences   (%) Beijing 2008 Finishing Time (h:min:s:ms) Differences   (%) Average Differences  (%) Men Gold 1:12:15.42 1st vs. 2nd 1.0 50:39.54 1st vs. 2nd 1.4 1:02:11.43 1st vs. 2nd 0.9 1st vs. 2nd 1.1 Silver 1:13:02.83 2nd vs. 3rd  0.3 51:21.54 2nd vs. 3rd  0.8 1:02.44.79 2nd vs. 3rd  1.0 2nd vs. 3rd  0.7 Bronze 1:13:17.54 1st vs. 3rd  1.4 51:47.87 1st vs. 3rd  2.2 1:03:21.11 1st vs. 3rd  1.8 1st vs. 3rd  1.8 Women Gold 44:26.42 1st vs. 2nd 0.2 37:34.82 1st vs. 2nd 0.7 34:51.72 1st vs. 2nd 1.2 1st vs. 2nd 0.7 Silver 44:31.97 2nd vs. 3rd  0.2 37:50.29 2nd vs. 3rd  0.3 35:16.01 2nd vs. 3rd  1.6 2nd vs. 3rd  0.7 Bronze 44:37.80 1st vs. 3rd  0.4 37:57.35 1st vs. 3rd  1.0 35:50.99 1st vs. 3rd  2.7 1st vs. 3rd  1.4 Olympic road cycling time trial results were reviewed and adapted from: https://www.olympic.org/cycling-road/individual-time-trial-men and https://www.olympic.org/cycling-road/individual-time-trial-women on October 8th 2017. 92  5.2.2 Future research on the diagnosis of EIB using the EVH challenge The high prevalence of bronchodilation in 81% of all athletes assessed in Chapter 4 made it difficult to answer the question of (1) whether an FI of 10% - 15% is a normative airway response to EVH, and (2) whether a shift of the cutoff distinguishing between an EVH+ and an EVH- test from 10% - 15% would decrease the rate of false-positives. Upon first sight, it may look like a shift from a 10% to a 15% cutoff would reduce the rate of false-positives based on the decreased number of 9, 10, 13, and 16 EVH+ tests to 2, 3, 3, and 10 EVH+ tests using FIA, FIB, FIC, and FID, respectively. However, the exercise-induced bronchodilation following the time trial was independent of the FI calculations, indicating that there was no relationship between the changes in FEV1 due to EVH and the time trial exercise. Therefore, before answering the question of whether an increase of the EVH+ cutoff serves to decrease the number of false-positives, further research comparing the bronchoconstriction stimulus induced by the EVH challenge and exercise protocols is needed. As mentioned in section 1.1.2., symptoms are poor predictors of the presence or the severity of EIB,1,14 which poses a challenge to the diagnosis of EIB.128 Assessing the correlation between markers of inflammation and the FI of an EVH challenge may provide further insight into the nature of a normative response to an EVH challenge is.105   Further research is needed to compare the voluntary hyperpnea asked of athletes in the EVH challenge to whole-body exercise. We agree with a previous suggestion by Price et al.105 to compare the bronchoconstriction stimulus generated by VEEVH-target to the bronchoconstriction stimulus of a whole-body exercise protocol that matches VEEVH-target. Furthermore, we recommend investigating mechanical respiratory differences between the EVH and the exercise 93  challenge with matching VEs. Multiple studies have shown differences in WOB, EELV, and EILV between whole body exercise and voluntary hyperpnea;118,119,129 however, it is unknown how pronounced these differences are in a six-minute protocol and whether these factors affect the dehydration of the airway surface liquid via changes in airflows (laminar vs. turbulent flow) and thus, the bronchoconstriction potential.   Having demonstrated a wide range of EVH challenge intensities when normalizing VEEVH-target by VEGXT in this thesis, further work is needed to assess the calculation of VEEVH-target as a percent of VEGXT, as previously suggested by Spiering et al.113 in an attempt to further standardize and individualize the EVH challenge. This could be particularly meaningful in the diagnosis of EIB in athletes with either very low or very high percent predicted values for FEV1.  Lastly, Hargreave and Nair116 highlight that variable airflow limitations are characteristic for asthma, a condition that shares symptoms with EIB. Variability in the degree of airflow limitation has also been shown in athletes with mild EIB,41,51 which makes the diagnosis of EIB difficult when relying on spirometry and FEV1. The accuracy and reliability of spirometry depends on technique,84 which can be a challenge for athletes, particularly when fatigue sets in after six minutes of voluntary hyperpnea. Further research on the diagnosis of EIB using alternative methods when assessing lung function is needed. Dickinson et al.130 assessed the sensitivity of forced expiratory flow at 50% of vital capacity (FEF50) in the diagnosis of EIB. In a previous study in children, expiratory flow at mid lung volumes was more sensitive to airway obstruction in the small airways than FEV1.131,132 In contrast to the hypothesis, FEF50 was less sensitive in the diagnosis of EIB compared to FEV1 using the EVH and exercise challenge in 94  adult athletes.130 Dickinson et al.130 concluded that expiratory airflow is just as likely to be restricted in the larger airways as in the smaller airways in EIB; therefore, using FEV1 as an index of function for the larger and the smaller airways of the lung is appropriate.  An alternative method of assessing airway dysfunction in the diagnosis of EIB could be impulse oscillometry (IOS). By applying sound waves to rapidly detect airway changes, IOS requires only normal tidal breathing from the test taker.133 Sound waves are superimposed on tidal breathing, and respiratory impedance is calculated by pressure and volume changes caused by the impulses during the measurement. When respiratory impedance values are expressed over a range of impulse frequencies, the detection of the site of airway obstruction can be performed. As a non-invasive, effort-independent method, IOS has emerged as a method to measure lung function in children in whom reliable spirometry is difficult to obtain. Impulse oscillometry has been compared to traditional spirometry and FEV1 in bronchoprovocation testing using an exercise protocol134 and the EVH protocol,135,136 but conflicting results require further research. Besides the reduced degree of cooperation during tidal breathing from the test taker, IOS appears not to cause respiratory fatigue, which is a known disadvantage of spirometry, in addition to the high degree of effort and motivation asked from test takers. Evans et al135 found a high correlation between spirometry and IOS when assessing the decrease in lung function following an EVH and an exercise challenge. In contrast to spirometry, IOS detected a difference in the degree of peak response to EVH when compared to exercise, suggesting that IOS may be a more sensitive measure of change in airway function.135 In contrast, a more recent study tested 16/94 athletes positive based on an FEV1 ≥ 10% and 17/94 athletes positive based on IOS (i.e. ≥ 50% decrease in the resistance at 5 Hz); however, only nine athletes met both diagnostic thresholds.136 Further research is needed to evaluate the use of IOS in the diagnosis of EIB in adult athletes. 95  References 1. Parsons JP, Hallstrand TS, Matronarde JG, et al. An Official American Thoracic Society clinical practice guideline: Exercise-induced bronchoconstriction. Am J Respir Crit Care Med. 2013;187(9):1016–1027. 2. Kippelen P, Anderson SD. Pathogenesis of exercise-induced bronchoconstriction. Immunol Allergy Clin. 2013;33(3):299–312. 3. McCallister JW, Mastronarde JG. Sex differences in asthma. J Asthma. 2008;45(10):853–861. 4. Kynyk JA, Mastronarde JG, McCallister JW. Asthma, the sex difference. Curr Opin Pulm Med. 2011;17(1):6–11. 5. Zein JG, Erzurum SC. Asthma is different in women. Curr Allergy Asthma Rep. 2015;15(6):28. 6. Becklake MR, Kauffmann F. Gender differences in airway behaviour over the human life span. Thorax. 1999;54(12):1119–1138. 7. Langdeau J-B, Day A, Turcotte H, Boulet L-P. Gender differences in the prevalence of airway hyperresponsiveness and asthma in athletes. Respir Med. 2009;103(3):401–406. 8. Johansson H, Norlander K, Berglund L, et al. Prevalence of exercise-induced bronchoconstriction and exercise-induced laryngeal obstruction in a general adolescent population. Thorax. 2015;70(1):57–63. 9. De Baets F, Bodart E, Dramaix-Wilmet M, et al. Exercise-induced respiratory symptoms are poor predictors of bronchoconstriction. Pediatr Pulmonol. 2005;39(4):301–305. 10. Busquets RM, Antó JM, Sunyer J, Sancho N, Vall O. Prevalence of asthma-related symptoms and bronchial responsiveness to exercise in children aged 13-14 years in Barcelona, Spain. Eur Respir J. 1996;9(10):2094–2098. 11. Polverino F, Matteis M, Spaziano G, et al. Effects of sex hormones on bronchial reactivity during the menstrual cycle. Eur Respir J. 2014;44(Suppl 58):P932. 12. Oguzulgen IK, Turktas H, Erbas D. Airway inflammation in premenstrual asthma. J Asthma. 2002;39(6):517-522. 13. Matteis M, Polverino F, Spaziano G, Roviezzo F, Santoriello C, Sullo N, Bucci MR, Rossi F, Polverino M, Owen CA, D'Agostino B. 2014. Effects of sex hormones on bronchial reactivity during the menstrual cycle. 2014;14:108-116. 96  14. Weiler JM, Brannan JD, Randolph CC, et al. Exercise-induced bronchoconstriction update-2016. J Allergy Clin Immunol. 2016.;138(5):1292-1295.e36. 15. Fitch KD. An overview of asthma and airway hyper-responsiveness in Olympic athletes. Br J Sports Med. 2012;46(6):413-416. 16. Naftali S, Rosenfeld M, Wolf M, Elad D. The air-conditioning capacity of the human nose. Ann Biomed Eng. 2005;33(4):545-553. 17. Cole P. Some aspects of temperature, moisture and heat relationships in the upper respiratory tract. J Laryngol Otol. 1953;67(8):449-456. 18. Inelstedt S, Ivstam B. Study in the humidifying capacity of the nose. Acta Oto-Laryngologica. 1951;39(4):286-290. 19. Proctor DF, Lundqvist G. Clearance of inhaled particles from the human nose. Arch Intern Med. 1973;131(1):132-139. 20. Bennett WD, Zeman KL, Jarabek AM. Nasal contribution to breathing with exercise: effect of race and gender. J Appl Physiol. 2003;95(2):497-503. 21. Cole P. Further consideration on the conditioning of respiratory air. J Laryngol Otol. 1953;67:669–681. 22. Svensson S, Olin AC, Hellgren J. Increased net water loss by oral compared to nasal expiration in healthy subjects. Rhinology. 2006;44(1):74-77. 23. Rundell KW, Sue Chu M. Air Quality and Exercise-Induced Bronchoconstriction in Elite Athletes. Immunol Allergy Clin North Am. 2013;33(3):409-421. 24. Anderson SD, Schoeffel RE, Follet R, Perry CP, Daviskas E, Kendall M. Sensitivity to heat and water loss at rest and during exercise in asthmatic patients. Eur J Respir Dis. 1982;63(5):459-471. 25. Anderson SD, Kippelen P. Exercise-induced bronchoconstriction: pathogenesis. Curr Allergy Asthma Rep. 2005;5(2):116-122. 26. McFadden ER Jr. Hypothesis: exercise-induced asthma as a vascular phenomenon. Lancet. 1990;335(8694):880-883. 27. Freed AN, Davis MS. Hyperventilation with dry air increases airway surface fluid osmolality in canine peripheral airways. Am J Respir Crit Care Med. 1999;159(4):1101-1107. 28. Davis MS, Daviskas E, Anderson SD. Airway surface fluid desiccation during isocapnic hyperpnea. J Appl Physiol. 2003;94(6):2545-2547. 97  29. Eschenbacher WL, Moore TB, Lorenzen TJ, Weg JG, Gross KB. Pulmonary responses of asthmatic and normal subjects to different temperature and humidity conditions in an environmental chamber. Lung. 1992;170(1):51-62. 30. Aitken ML, Marini JJ, Williams D. Effect of heat delivery and extraction on airway conductance in normal and in asthmatic subjects. Am Rev Respir Dis. 1985;(131):357-361. 31. Tschumperlin DJ, Drazen JM. Chronic effects of mechanical force on airways. Ann Rev Physiol. 2006;68(1):563-583. 32. Boulet L-P. Cough and upper airway disorders in elite athletes: A critical review. Br J Sports Med. 2012;46(6):417-421. 33. Chowdhary R, Singh V, Tattersfield AE, Sharma SD, Kar S, Gupta AB. Relationship of flow and cross-sectional area to frictional stress in airway models of asthma. J Asthma. 2009;36(5):419-426. 34. Jensen A, Atileh H, Suki B, Ingenito EP, Lutchen KR. Selected contribution: Airway caliber in healthy and asthmatic subjects: effects of bronchial challenge and deep inspirations. J Appl Physiol. 2001;91(1):506-515. 35. Anderson SD, Kippelen P. Assessment of EIB: What you need to know to optimize test results. Immunol Allergy Clinf North Am. 2013;33(3):363-380. 36. Sue Chu M, Brannan JD, Anderson SD, Chew N, Bjermer L. Airway hyperresponsiveness to methacholine, adenosine 5-monophosphate, mannitol, eucapnic voluntary hyperpnoea and field exercise challenge in elite cross-country skiers. Br J Sports Med. 2010;44(11):827-832. 37. Crapo RO, Casaburi R, Coates AL, et al. Guidelines for methacholine and exercise challenge testing-1999. Am J Respir Crit Care Med. 2000;161(1):309-329. 38. Carlsen K-H, Engh G, Mørk M. Exercise-induced bronchoconstriction depends on exercise load. Respi Med. 2000;94(8):750-755. 39. World Anti-Doping Agency. The Prohibited List 2017. https://www.wada-ama.org/sites/default/files/resources/files/2016-09-29_-_wada_prohibited_list_2017_eng_final.pdf. Accessed October 12th, 2017. 40. Weiler JM, Bonini S, Coifman R, Craig T. American Academy of Allergy, Asthma & Immunology work group report: Exercise-induced asthma. J Allergy Clin Immunol. 2007.119(6):1349-1358.   98  41. Anderson SD, Pearlman DS, Rundell KW, et al. Reproducibility of the airway response to an exercise protocol standardized for intensity, duration, and inspired air conditions, in subjects with symptoms suggestive of asthma. Respir Res. 2010;11(1):120-132 42. Hurwitz KM, Argyros GJ, Roach JM, Eliasson AH, Phillips YY. Interpretation of eucapnic voluntary hyperventilation in the diagnosis of asthma. Chest. 1995;108(5):1240-1245. 43. Phillips YY, Jaeger JJ, Laube BL, Rosenthal RR. Eucapnic voluntary hyperventilation of compressed gas mixture. A simple system for bronchial challenge by respiratory heat loss. Am Rev Respir Dis. 1985;131(1):31-35. 44. Argyros GJ. Eucapnic voluntary hyperventilation as a bronchoprovocation technique. Chest. 1996;109(6):1520-1524. 45. International Olympic Committee. Guidelines for beta2-adrenoceptor agonists and the Olympic Games in Beijing. https://stillmed.olympic.org/media/Document%20Library/OlympicOrg/Games/Summer-Games/Games-Beijing-2008-Olympic-Games/Anti-doping-and-Medical-Rules/Beta2-adrenoceptor-agonists-Guidelines-Beijing-2008.pdfAccessed October 10th, 2017. 46. Anderson SD, Argyros GJ, Magnussen H, Holzer K. Provocation by eucapnic voluntary hyperpnoea to identify exercise induced bronchoconstriction. Br J Sports Med. 2001;35(5):344-347. 47. Price OJ, Ansley L, Levai IK, et al. Eucapnic voluntary hyperpnea testing in asymptomatic athletes. Am J Respir Crit Care Med. 2016;193(10):1178-1180. 48. Backer V, Sverrild A, Porsbjerg C. Treatment of exercise-induced bronchoconstriction. Immunol Allergy Clin of North Am. 2013;33(3):347-362. 49. Stickland MK, Rowe BH, Spooner CH, Vandermeer B, Dryden DM. Effect of Warm-up exercise on exercise-induced bronchoconstriction. Med Sci Sports Exerc. 2012;44(3):383-391. 50. Fitch KD. The enigma of inhaled salbutamol and sport: Unresolved after 45 years. Drug Test Anal. 2017;9(7):977-982. 51. Hallstrand TS, Kippelen P, Larsson J, et al. Where to from here for exercise-induced bronchoconstriction. Immunol Allergy Clin. 2013;33(3):423-442. 52. Davis E, Loiacono R, Summers RJ. The rush to adrenaline: drugs in sport acting on the β‐adrenergic system. Br J Pharmacol. 2008;154(3):584-597.  99  53. Minakata Y, Suzuki S, Grygorczyk C, Dagenais A, Berthiaume Y. Impact of β-adrenergic agonist on Na+ channel and Na+-K+-ATPase expression in alveolar type II cells. Am J Physiol. 1998;275(2):L414-L422. 54. Johnson MD, Bao H-F, Helms MN, et al. Functional ion channels in pulmonary alveolar type I cells support a role for type I cells in lung ion transport. PNAS. 2006;103(13):4964-4969. 55. Broadley KJ. Beta-adrenoceptor responses of the airways: for better or worse? Eur J Pharmacol. 2006;533(1-3):15-27. 56. Hanania NA, Moore RH. Anti-inflammatory activities of beta2-agonists. Curr Drug Targets Inflamm Allergy. 2004;3(3):271-277. 57. Carlsen K-H, Anderson SD, Bjermer L, et al. Exercise‐induced asthma, respiratory and allergic disorders in elite athletes: epidemiology, mechanisms and diagnosis: Part I of the report from the Joint Task Force of the European Respiratory Society (ERS) and the European Academy of Allergy and Clinical Immunology (EAACI) in cooperation with GA2LEN. Allergy. 2008;63(4):387–403. 58. Pluim B, de Hon O, Staal J, Limpens J. Beta2-agonists and physical performance: A systematic review and meta-analysis of randomized controlled trials. Sports Med. 2011;41(1):39-57. 59. Kinderman. Do inhaled beta2-agonists have an ergogenic potential in non-asthmatic competitive athletes? Sports Med. 2007;37(2):95-102. 60. Koch S, MacInnis MJ, Sporer BC, Rupert JL, Koehle MS. Inhaled salbutamol does not affect athletic performance in asthmatic and non-asthmatic cyclists. Br J Sports Med. 2013;49(1):51-55. 61. Sporer BC, Sheel AW, McKenzie DC. Dose response of inhaled salbutamol on exercise performance and urine concentrations. 2008;40(1):149-157. 62. Meeuwisse WH, McKenzie DC, Hopkins SR, Road JD. The effect of salbutamol on performance in elite nonasthmatic athletes. Med Sci Sports Exerc. 1992;24(10):1161-1166. 63. Harms CA, Rosenkranz S. Sex differences in pulmonary function during exercise. Med Sci Sports Exerc. 2008;40(4):664-668. 64. Sheel AW, Dominelli PB, Molgat-Seon Y. Revisiting dysanapsis: sex‐based differences in airways and the mechanics of breathing during exercise. Exp Physiol. 2016;101(2):213-218. 65. Sheel AW, Richards JC, Foster GE, Guenette JA. Sex differences in respiratory exercise physiology. Sports Med. 2004;34(9):567-579. 100  66. Green M, Mead J, Turner JM. Variability of maximum expiratory flow-volume curves. J Appl Physiol. 1974;37(1):67-74. 67. Mead J. Dysanapsis in normal lungs assessed by the relationship between maximal flow, static recoil, and vital capacity. Am Rev Respir Disease. 1980;121(2):339-342. 68. Martin TR, Castile RG, Fredberg JJ, Wohl ME, Mead J. Airway size is related to sex but not lung size in normal adults. J Appl Physiol. 1987;63(5):2042-2047. 69. Sheel AW, Guenette JA, Yuan R, et al. Evidence for dysanapsis using computed tomographic imaging of the airways in older ex-smokers. J Appl Physiol. 2009;107(5):1622-1628. 70. Harms CA. Does gender affect pulmonary function and exercise capacity? Respir Physiol Neurobiol. 2006;151(2-3):124-131. 71. Guenette JA, Witt JD, McKenzie DC. Respiratory mechanics during exercise in endurance‐trained men and women. J Physiol. 2007;581(3):1309-1322. 72. McClaran SR, Harms CA, Pegelow DF, Dempsey JA. Smaller lungs in women affect exercise hyperpnea. J Appl Physiol. 1998;84(6):1872-1881. 73. Dempsey JA, McKenzie DC, Haverkamp HC, Eldridge MW. Update in the understanding of respiratory limitations to exercise performance in fit, active adults. Chest. 2008;134(3):613-622. 74. Smith JR, Rosenkranz SK, Harms CA. Dysanapsis ratio as a predictor for expiratory flow limitation. Respir Physiol Neurobiol. 2014;198:25-31. 75. Dominelli PB, Molgat-Seon Y, Bingham D, et al. Dysanapsis and the resistive work of breathing during exercise in healthy men and women. J Appl Physiol. 2015:119(10):1105-1113. 76. Guenette JA, Querido JS, Eves ND, Chua R, Sheel AW. Sex differences in the resistive and elastic work of breathing during exercise in endurance-trained athletes. Am J Physiol Regul Integr Comp Physiol. 2009;297(1):R166-75. 77. Harms CA, Babcock MA, McClaran SR, et al. Respiratory muscle work compromises leg blood flow during maximal exercise. J Appl Physiol. 1997;82(5):1573-1583. 78. Dickinson J, Hu J, Chester N, Loosemore M, Whyte G. Acute impact of inhaled short acting b2-agonists on 5 km running performance. J Sports Sci Med. 2014;13(2):271-279. 79. Dickinson J, Molphy J, Chester N, Loosemore M, Whyte G. The ergogenic effect of long-term use of high dose salbutamol. Clin J Sport Med. 2014;24(6):474–481. 101  80. Kalsen A, Hostrup M, Bangsbo J, Backer V. Combined inhalation of beta2‐agonists improves swim ergometer sprint performance but not high‐intensity swim performance. Scand J Med Sci Sports. 2014;24(5):814-822. 81. Sheel AW, Guenette JA. Mechanics of breathing during exercise in men and women. Exerc Sport Sci Rev. 2008;36(3):128-134. 82. Beidleman BA, Rock PB, Muza SR, Fulco CS, Forte VA, Cymerman A. Exercise V˙e and physical performance at altitude are not affected by menstrual cycle phase. J Appl Physiol. 1999;86(5):1519-1526. 83. MacNutt MJ, De Souza MJ, Tomczak SE, Homer JL, Sheel AW. Resting and exercise ventilatory chemosensitivity across the menstrual cycle. J Appl Physiol. 2012;112(5):737-747. 84. Miller MR, Hankinson J, Brusasco V, et al. Standardisation of spirometry. Eur Respir J. 2005;26(2):319-338. 85. Borg G. Psychophysical scaling with applications in physical work and the perception of exertion. Scand J Work, Environ Health. 1990;16:55-58. 86. Johnson BD, Scanlon PD, Beck KC. Regulation of ventilatory capacity during exercise in asthmatics. J Appl Physiol. 1995;79(3):892-901. 87. Haverkamp HC, Dempsey JA, Miller JD, et al. Gas exchange during exercise in habitually active asthmatic subjects. J Appl Physiol. 2005;99(5):1938-1950. 88. Martineau L, Horan MA, Rothwell NJ, Little RA. Salbutamol, a beta 2-adrenoceptor agonist, increases skeletal muscle strength in young men. Clin Sci. 1992;83(5):615-621. 89. van Baak M, Mayer LHJ, Kempinski RES, Hartgens F. Effect of salbutamol on muscle strength and endurance performance in nonasthmatic men. Med Sci Sports Exerc. 2000;32(7):1300-1306. 90. Collomp K, Candau R, Collomp R, et al. Effects of acute ingestion of salbutamol during submaximal exercise. Int J Sports Med. 2000;21(07):480-484. 91. Sanchez AM, Collomp K, Carra J, et al. Effect of acute and short-term oral salbutamol treatments on maximal power output in non-asthmatic athletes. Eur J Appl Physiol. 2012;112(9):3251-3258. 92. Koch S, Karacabeyli D, Galts C, MacInnis MJ, Sporer BC, Koehle MS. Effects of inhaled bronchodilators on lung function and cycling performance in female athletes with and without exercise-induced bronchoconstriction. J Sci Med Sport. 2015;18(5):607-612. 102  93. Palmer KN. Muscle cramp and oral salbutamol. Br Med J. 1978;2(6140):833. 94. Lisi DM. Muscle spasms and creatine phosphokinase elevation following salbutamol administration. Eur Respir J. 1989;2(1):98. 95. Craig TJ, Smits W, Soontornniyomkiu V. Elevation of creatine kinase from skeletal muscle associated with inhaled albuterol. 1996;77(6):488-490. 96. Hostrup M, Kalsen A, Auchenberg M, Bangsbo J, Backer V. Effects of acute and 2‐week administration of oral salbutamol on exercise performance and muscle strength in athletes. Scand J Med Sci Sports. 2014;26(1):8-16. 97. Drazen JM, Boulet L-P, O’Byrne PM. Asthma and exercise-induced bronchoconstriction in athletes. N Engl J Med. 2015;372(7):641-648. 98. Vilozni D, Szeinberg A, Barak A, Yahav Y, Augarten A, Efrati O. The relation between age and time to maximal bronchoconstriction following exercise in children. Respir Med. 2009;103(10):1456-1460. 99. Batista C, Soares JM. Are former athletes more prone to asthma? J Asthma. 2013;50(4):403-409. 100. Rundell KW, Anderson SD, Sue Chu M, Bougault V, Boulet L-P. Air Quality and Temperature Effects on Exercise‐Induced Bronchoconstriction. Compr Physiol. 2015;2:579-610. 101. Holzer K, Anderson SD, Douglass J. Exercise in elite summer athletes: Challenges for diagnosis. J Allergy Clin Immunol. 2002;110(3):374-380. 102. Hull JHK, Ansley L, Garrod R, Dickinson JW. Exercise-induced bronchoconstriction in athletes-should we screen? Med Sci Sports Exerc. 2007;39(12):2117-2124. 103. Rosenthal RR. Simplified eucapnic voluntary hyperventilation challenge. J Allergy Clin Immunol. 1984;73(5 Pt 2):676-679. 104. Price OJ, Ansley L, Hull JH. Diagnosing exercise-induced bronchoconstriction with eucapnic voluntary hyperpnea: Is one test enough? J Allergy Clinical Immunol Pract. 2014;3(2):243-249. 105. Mastronarde J. Eucapnic voluntary hyperpnea testing in athletes. Am J Respir Crit Care Med. 2017;195(7):960-961. 106. Dickinson J, McConnell A, Whyte G. Diagnosis of exercise-induced bronchoconstriction: eucapnic voluntary hyperpnoea challenges identify previously undiagnosed elite athletes with exercise-induced bronchoconstriction. Br J Sports Med. 2011;45(14):1126-1131. 103  107. Ansley L, Kippelen P, Dickinson J, Hull JHK. Misdiagnosis of exercise-induced bronchoconstriction in professional soccer players. Allergy. 2012;67(3):390-395. 108. Weiler JM, Hallstrand TS, Parsons JP, et al. Improving screening and diagnosis of exercise-induced bronchoconstriction: A call to action. J Allergy Clin Immunol Practice. 2014;2(3):275–570.e7. 109. Parsons JP, Hallstrand TS, Mastronarde JG, et al. An Official American Thoracic Society Clinical Practice Guideline: Exercise-induced bronchoconstriction. Am J Respir Crit Care Med. 2013;187(9):1016-1027. 110. McFadden ER, Gilbert IA. Exercise-induced asthma. N Engl J Med. 1994;330(19):1362–1367. 111. Koch S, Ahn JR, Koehle MS. High-dose inhaled salbutamol does not improve 10-km cycling time trial performance. Med Sci Sports Exerc. 2015;47(11):2373-2379. 112. Anderson SD, Brannan JD. Methods for “indirect” challenge tests including exercise, eucapnic voluntary hyperpnea, and hypertonic aerosols. Clin Rev Allergy Immunol. 2003;24(1):27-54. 113. Spiering BA, Judelson DA, Rundell KW. An evaluation of standardizing target ventilation for eucapnic voluntary hyperventilation using FEV1. J Asthma. 2009;41(7):745-749. 114. Brummel NE, Mastronarde JG, Rittinger D, Philips G, Parsons JP. The clinical utility of eucapnic voluntary hyperventilation testing for the diagnosis of exercise-induced bronchospasm. J Asthma. 2009;46(7):683-686. 115. Dominelli PB, Render JN, Molgat-Seon Y, Foster GE, Romer LM, Sheel AW. Oxygen cost of exercise hyperpnoea is greater in women compared with men. J Physiol. 2015;593(8):1965-1979. 116. Hargreave FE, Nair P. The definition and diagnosis of Asthma. Clin Exp Allergy. 2009;39(11):1652-1658. 117. Barnes PJ, Brown MJ, Silverman M, Dollery CT. Circulating catecholamines in exercise and hyperventilation induced asthma. Thorax. 1981;36(6):435-440. 118. Dominelli PB, Render JN, Molgat-Seon Y, Foster GE, Sheel AW. Precise mimicking of exercise hyperpnea to investigate the oxygen cost of breathing. Respir Physiol Neurobiol. 2014;201:15-23. 119. Klas JV, Dempsey JA. Voluntary versus reflex regulation of maximal exercise flow: volume loops. Am Rev Respir Dis. 1989;139(1):50-56.  104  120. Blackie SP, Hilliam C, Village R, Paré PD. The time course of bronchoconstriction in asthmatics during and after isocapnic hyperventilation. Am Rev Respir Dis. 1990;142(5):1133-1136. 121. Fitch K. The world anti-doping code: can you have asthma and still be an elite athlete? Breathe. 2016;12(2):148-158. 122. Haase CB, Backer V, Kalsen A, Rzeppa S, Hemmersbach P, Hostrup M. The influence of exercise and dehydration on the urine concentrations of salbutamol after inhaled administration of 1600 µg salbutamol as a single dose in relation to doping analysis. Drug Test Anal. 2016;8(7)613-620. 123. Dickinson J, Hu J, Chester N, Loosemore M, Whyte G. Impact of ethnicity, gender, and dehydration on the urinary excretion of inhaled salbutamol with respect to doping control. Clin J Sport Med. 2014;24(6):482-489. 124. Elers J, Hostrup M, Pedersen L, et al. Urine and serum concentrations of inhaled and oral terbutaline. Int J Sports Med. 2012;33(12):1026-1033. 125. Wasserstein RL, Lazar NA. The ASA's statement on p-values: Context, process, and purpose. American Statistician. 2016;70(2):129-133. 126. Hopkins WG, Hawley JA, Burke LM. Design and analysis of research on sport performance enhancement. Med Sci Sports Exerc. 1999;31(3):472-485. 127. Kearney JT. Sport performance enhancement: design and analysis of research. Med Sci Sports Exerc. 1999;31(5):755-757. 128. Hull JH, Ansley L, Price OJ, Dickinson JW, Bonini M. Eucapnic voluntary hyperpnea: gold standard for diagnosing exercise-induced bronchoconstriction in athletes? Sports Med. 2016;46(8):1-11. 129. Johnson BD, Weisman IM, Zeballos RJ, Beck KC. Emerging concepts in the evaluation of ventilatory limitation during exercise: the exercise tidal flow-volume loop. Chest. 1999;116(2):488-503. 130. Dickinson JW, Whyte GP, McConnell AK, Nevill AM, Harries MG. Mid-expiratory flow versus FEV1 measurements in the diagnosis of exercise induced asthma in elite athletes. Thorax. 2006;61(2):111-114. 131. Lebecque P, Kiakulanda P, Coates AL. Spirometry in the asthmatic child: is FEF25-75 a more sensitive test than FEV1/FVC? Pediatr Pulmonol. 1993;16(1):19-22. 132. Bar-Yishay E, Amirav I, Goldberg S. Comparison of maximal midexpiratory flow rate and forced expiratory flow at 50% of vital capacity in children. Chest. 2003;123(3):731-735. 105  133. Bickel S, Popler J, Lesnick B, Eid N. Impulse oscillometry: interpretation and practical applications. Chest. 2014;146(3):841-847. 134. Evans TM, Rundell KW, Beck KC, Levine AM, Baumann JM. Airway narrowing measured by spirometry and impulse oscillometry following room temperature and cold temperature exercise. Chest. 2005;128(4):2412-2419. 135. Evans TM, Rundell KW, Beck KC, Levine AM, Baumann JM. Impulse oscillometry is sensitive to bronchoconstriction after eucapnic voluntary hyperventilation or exercise. J Asthma. 2006;43(1):49-55. 136. Price OJ, Ansley L, Bikov A, Hull JH. The role of impulse oscillometry in detecting airway dysfunction in athletes. J Asthma. 2016;53(1):62-68.  106  Appendix Appendix: Supplemental material for Chapter 4: Evaluation of the diagnostic criteria of the eucapnic voluntary hyperpnea test in trained athletes.  Supplemental Table S1: Baseline FEV1, and fall indices of the four calculation methods including time trial spirometry for male and female athletes.  ID FEV1 BL (L) FIA (%) FIB (%) FIC (%) FID  (%) FITT (%) F1 4.2 -4.1 -5.3 -5.3 -6.6 +7.2 F3* 3 -10.3 -15.7 -14.7 -16.1 -1.7 F7 3.9 -6.9 -7.4 -7.4 -9.6 +3.5 F8 3.4 -4.4 -5.3 -5.3 -6.6 -1.2 F9 3.9 -10.6 -13.7 -11.9 -5.9 +3.3 F11 3.8 -1.6 -2.9 -24.7 -38.0 +2.2 F12 4.3 -2.8 -2.8 -3.0 -5.3 +1.2 F13 4.9 -3.9 -5.5 -4.9 -8.1 +4.4 F14 3.6 -4.2 -5.1 -6.2 -6.4 +3.8 F15 3.8 -5.9 -5.9 -6.4 -8.9 +5.9 F16 3.4 -5.1 -5.1 -12.8 -17.6 +8.6 F17 4.1 -15.3 -21.5 -21.5 -29.5 +4.3 F18 4.0 -3.3 -3.3 -3.3 -4.0 +4.6 F19 2.8 -3.6 -5.0 -5.0 -6.2 +6.2 F20 3.7 -7.1 -8.4 -7.6 -13.0 -2.9 F21 3.6 -4.5 -8.7 -8.7 -12.6 +5.3 F24 5.5 -4.5 -4.7 -4.7 -8.1 +1.1 F25 3.6 -3.1 +5.3 -4.7 -6.2 +7.4 F26* 4.0 -10.3 -10.3 -11.0 -16.4 +8.2 M4 6.1 -4.9 -7.2 -5.1 -8.3 -9.1 M5* 4.8 -8.6 -9.7 -9.7 -13.3 +7.0 M6 4.6 -2.9 -2.9 -2.9 -3.7 +3.8 M7* 4.0 -14.4 -14.9 -14.9 -18.5 +2.5 M10 5.4 -9.5 -14.3 -14.3 -20.3 -7.9 M11 4.9 -3.7 -4.7 -4.7 -5.5 +31.9 M12 3.9 +6.7 +6.7 +6.4 +7.7 +1.2 M14 5.3 -1.1 -2.3 -1.3 -1.9 +1.1 M15 5.2 -19.9 -19.9 -19.9 -24.9 +2.0 M16 5.1 -3.0 -4.9 -10.0 -11.9 +2.2 107  ID FEV1 BL (L) FIA (%) FIB (%) FIC (%) FID  (%) FITT (%) M18 5.1 -10.0 -10.4 -10.4 -13.2 +6.6 M19 4.9 -10.6 -11.8 -11.1 -15.2 +3.2 M20 3.8 -2.1 -1.3 -2.1 -2.7 +1.1 M22* 4.1 -10.8 -13.8 -13.8 -16.7 -2.2 M24 5.1 -3.5 -6.3 -6.3 -8.7 +25.5 M25 4.9 -4.9 -4.3 -4.9 -6.1 +3.9 M26 5.7 -3.3 -3.3 -4.2 -4.8 +2.1 M27 5.1 -6.3 -8.2 -7.2 -10.0 -5.5 AVG, average; FEV1 BL, forced expiratory volume in 1 second at baseline; FI, fall index.  *indicated athlete with a previous diagnosis of asthma or EIB.  

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