Education, Faculty of
Kinesiology, School of
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UBCV
Harris, Owen Drake
Master of Science - MSc
University of British Columbia
Ground-level ozone (O₃) is a common airborne pollutant well recognized to impair pulmonary function and cause negative respiratory symptoms when inhaled. Due to a wide range of inter-individual variability in responses, a lack of consensus as to whether O₃ exposure limits exercise capability exists. Healthy, well-trained male (n=13) and female (n=7) endurance athletes were recruited to complete a double-blinded, randomized crossover study to address the research question. Participants were screened for inclusion based on a cycling maximal oxygen consumption (V̇O₂max) test (Visit 1), before resting exposure to 750 parts per billion (ppb) O₃ (Visit 2) to assess pulmonary function changes. During experimental trials (Visit 3 & 4), participants completed submaximal and maximal cycling exercise protocols exposed to both O₃ (170 ppb) and room air (RA, <10 ppb O₃). Results (n=20, V̇O₂max = 64.1 ± 7.0 ml/kg/min) indicated that pulmonary function was significantly reduced and development of adverse respiratory symptoms was increased following both resting and exercise O₃ exposure. No differences between conditions in ventilatory pattern or oxygen consumption (V̇O₂) were observed throughout submaximal exercise bouts. Paired t-tests comparing the last minute of a time-to-exhaustion (TTE) test at 105% of peak power showed lower relative V̇O₂ (p=0.003), tidal volume (p=0.007) and minute ventilation (p=0.047) during O₃ compared to RA conditions, with insignificant trends towards lower exercise time (p=0.092). As differences in relative V̇O₂ (p<0.001) and tidal volume (p=0.001) also existed when comparing each participant’s last shared minute between conditions, a respiratory limitation of oxygen transport to the exercising muscle groups during maximal exercise seems likely. A clearer understanding of the mechanisms that impact oxygen consumption during O₃ exposure and exercise is critical to maximizing performance in athletic events exposed to high levels of pollution.
INVESTIGATING THE SUSCEPTIBILITY OF CARDIORESPIRATORY RESPONSES TO OZONE DURING CYCLING EXERCISE by OWEN DRAKE HARRIS B.H.K., The University of British Columbia Okanagan, 2021 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Kinesiology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2023 © Owen Drake Harris, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Investigating the susceptibility of cardiorespiratory responses to ozone during cycling exercise submitted by Owen Drake Harris in partial fulfilment of the requirements for the degree of Master of Science in Kinesiology Examining Committee: Dr. Michael Koehle, Professor, School of Kinesiology, UBC Supervisor Dr. William Sheel, Professor, School of Kinesiology, UBC Supervisory Committee Member Dr. Valérie Bougault, Associate Professor, Sciences du Sport, Université Côte d'Azur Supervisory Committee Member iii Abstract Ground-level ozone (O3) is a common airborne pollutant well recognized to impair pulmonary function and cause negative respiratory symptoms when inhaled. Due to a wide range of inter-individual variability in responses, a lack of consensus as to whether O3 exposure limits exercise capability exists. Healthy, well-trained male (n=13) and female (n=7) endurance athletes were recruited to complete a double-blinded, randomized crossover study to address the research question. Participants were screened for inclusion based on a cycling maximal oxygen consumption (?̇?O2max) test (Visit 1), before resting exposure to 750 parts per billion (ppb) O3 (Visit 2) to assess pulmonary function changes. During experimental trials (Visit 3 & 4), participants completed submaximal and maximal cycling exercise protocols exposed to both O3 (170 ppb) and room air (RA, <10 ppb O3). Results (n=20, ?̇?O2max = 64.1 ± 7.0 ml/kg/min) indicated that pulmonary function was significantly reduced and development of adverse respiratory symptoms was increased following both resting and exercise O3 exposure. No differences between conditions in ventilatory pattern or oxygen consumption (?̇?O2) were observed throughout submaximal exercise bouts. Paired t-tests comparing the last minute of a time-to-exhaustion (TTE) test at 105% of peak power showed lower relative ?̇?O2 (p=0.003), tidal volume (p=0.007) and minute ventilation (p=0.047) during O3 compared to RA conditions, with insignificant trends towards lower exercise time (p=0.092). As differences in relative ?̇?O2 (p<0.001) and tidal volume (p=0.001) also existed when comparing each participant’s last shared minute between conditions, a respiratory limitation of oxygen transport to the exercising muscle groups during maximal exercise seems likely. A clearer understanding of the mechanisms that impact oxygen consumption during O3 exposure and exercise is critical to maximizing performance in athletic events exposed to high levels of pollution. iv Lay Summary Air pollution, and particularly ground-level ozone (O3) can cause health problems when inhaled to due to interactions with the lungs and conducting airways. However, the ways which O3 may impact the ability to perform exercise in highly-trained athletes is currently unknown, largely due to a wide range in responses amongst individuals. Twenty healthy endurance athletes were recruited to perform resting, submaximal, and maximal exercise in both O3 and room air to examine the impacts of real-world levels of O3 exposure. Results showed that pulmonary function was reduced, development of respiratory symptoms was increased, and that participants adopted a more inefficient breathing style during exercise with O3 exposure. It seems likely that O3 exposure limits the efficiency of breathing, reducing the transport of oxygen to the working muscles and makes exercise more challenging. Understanding the mechanisms that cause these reductions is key to athletes performing exercise in areas of high pollution. v Preface This thesis is the original intellectual work of the author, Owen Drake Harris, under the supervision of Dr. Michael Koehle. All work presented henceforth was conducted in the Environmental Physiology Laboratory at the University of British Columbia, Point Grey campus. The study and methods received ethics approval from the University of British Columbia’s Clinical Research Ethics Board (certificate #: H22-01389). The author, Owen Drake Harris, served as the lead investigator and was responsible for all major areas of concept formation, experimental design, ethical approval, participant recruitment, data collection, data analysis, and manuscript composition. Joey Cheng, Sacha Schimmelpenninck, Patric Gonçalves, Bennett Stothers, Andy Hung, Owen Payne, and Steve Busch assisted with data collection by operating the O3 chamber to maintain double-blinding of participants and the primary researcher. Other members of the Environmental Physiology Laboratory (Gareth Sandford, Assaf Yogev, Jem Arnold) assisted with project design and analysis. Supervisory committee Dr. Bill Sheel and Dr. Valérie Bougault provided guidance regarding study design, analysis, and interpretation of results. The principal investigator, Dr. Michael Koehle, provided supervisory oversight on all aspects of the study. vi Table of Contents Abstract........................................................................................................................................ iii Lay Summary............................................................................................................................... iv Preface.............................................................................................................................................v Table of Contents......................................................................................................................... vi List of Tables.............................................................................................................................. viii List of Figures............................................................................................................................... ix List of Abbreviations.................................................................................................................... x Acknowledgements...................................................................................................................... xi Dedication.................................................................................................................................... xii Chapter 1: Introduction…………………………………………………………………... 1 1.1 Background………………………………………………………………. 1 1.2 Research Question…………………………...…………………………... 7 1.3 Hypothesis…………………………………………………………...…… 8 Chapter 2: Methods………………………………………………………………………. 9 2.1 Sample and Recruitment………………………………………................. 9 2.2 Sample Size Calculation……………………………………………..…. 12 2.3 Measures/Instruments…………………………………………………... 12 2.4 Study Design……………………………………………….…………… 16 2.5 Procedures………………………….…………………………………… 16 2.6 Data Analysis………………………………………..………………….. 21 Chapter 3: Results………………………………………………………...…………….. 22 3.1 Participant Recruitment………………………………………………… 22 vii 3.2 Participant Characteristics……………………………………………… 22 3.3 O3 Concentrations……………………….……………………………… 23 3.4 Resting O3 Pulmonary Function and Symptom Responses…….………. 23 3.5 Exercise Intensity………………………………….…………….……… 28 3.6 25-min Warmup Exercise Response……………………………………. 29 3.7 30-min Constant Work Rate Bout Exercise Response…………………. 30 3.8 Pulmonary Function Response to Exercise…………………….…….…. 35 3.9 Symptom Development with Exercise……………………………….…. 39 3.10 Time-to-Exhaustion Test Exercise Response..…………………………. 44 Chapter 4: Discussion…………………………...……………………………………… 53 4.1 Summary………………………………….…………………………….. 53 4.2 Response during Submaximal Exercise……………….………………... 53 4.3 Response during Maximal Exercise…………………… ………………. 56 4.4 Symptom Development with Exercise…………………………….……. 61 4.5 Pulmonary Function Changes with Exercise…………………………… 63 4.6 Resting O3 Pulmonary Function Response………………...…………… 66 4.7 Individual Susceptibility to Responses…………………………………. 70 4.8 Study Limitations………………………………………………….……. 71 Chapter 5: Significance…………………………………………………………….…… 72 5.2 Potential Impact………………………………………………………… 72 5.3 Future Directions……………………………………………………….. 72 Chapter 6: Conclusion………………………………………….…………………...…... 74 References……………………………………………………………………………………… 75 viii List of Tables Table 2.1. Inclusion and exclusion criteria…………………………………………………...….. 9 Table 3.1. Changes in metabolic variables during a 25 min warmup cycling bout at 90% of first lactate threshold………………………………………………………………………………… 30 Table 3.2. Changes in metabolic variables during a 30 min warmup cycling bout at 90% of second lactate threshold………………………………………………………………………… 35 Table 3.3. Changes in metabolic variables during the last minute of a time-to-exhaustion cycling bout at 105% of peak power.…………………………………………………………………… 49 Table 3.3. Changes in metabolic variables during the last shared minute of a time-to-exhaustion cycling bout at 105% of peak power.…………………………………………………………… 52 ix List of Figures Figure 2.1. Example of instrumentation during experimental visits……………………………. 20 Figure 2.2. Ozone concentration control setup………………………………………...……….. 21 Figure 3.1. Change in FVC (L) and FEV1 (L) following a 2-hour exposure to 750 ppb O3….... 26 Figure 3.2. Change in FEV1/FVC (%), PEFR (L/s), and FEF25-75 (L/s) following a 2-hour exposure to 750 ppb…………………………………………………………………………….. 27 Figure 3.3. Change in RER throughout 30-minute constant work-rate bout at 90% of LT2…… 33 Figure 3.4. Change in breathing frequency throughout a 30-minute constant work-rate bout at 90% of LT2…………………………………………………………………………………...… 34 Figure 3.5. Change in tidal volume throughout a 30-minute constant work-rate bout at 90% of LT2……………………………………………………………………………………………… 34 Figure 3.6. Change in FVC (L), FEV1 (L), FEV1/FVC (%) and PEFR (L/s) with O3 exposure and exercise…………...………………………………………………………………………….…. 38 Figure 3.7. Change in FEF25-75 (L/s) with O3 exposure and exercise…………………………… 39 Figure 3.8. Change in symptom development with O3 exposure and exercise………………..... 43 Figure 3.9. Change in dyspnea development with O3 exposure and exercise……………...…... 44 Figure 3.10. Change in relative ?̇?O2 and VCO2 in the last minute of the TTE………………… 46 Figure 3.11. Change in RER, ?̇?E, FB, and VT in the last minute of the TTE……………....…… 47 Figure 3.12. Change in ?̇?E/?̇?O2, ?̇?E/VCO2, HR, and TTE in the last minute of the TTE….….. 48 Figure 3.13. Change in relative ?̇?O2, VCO2, RER, and ?̇?E in the last shared minute of the TTE. ………………………………………………………………………………………..…………. 50 Figure 3.16. Change in FB, VT, ?̇?E/?̇?O2, and ?̇?E/VCO2 in the last shared minute of the TTE.... 51 Figure 3.17. Change in HR in the last shared minute of the TTE………………………………. 52 x List of Abbreviations ATS American Thoracic Society BLa Blood lactate CO2 Carbon dioxide CWR Constant work-rate FB Breathing frequency FEF25-75 Forced expiratory flow at 25–75% of FVC FEV1 Forced expired volume in 1-second FEV1/FVC Ratio of FEV1 to FVC FVC Force vital capacity GXT Graded exercise test MLSS Maximal lactate steady state NO2 Nitrogen dioxide NOx Nitrogen oxides O2 Oxygen O3 Ozone OBLA Onset of blood lactate accumulation PEFR Peak expiratory flow rate PFT Pulmonary function test Ppb Parts per billion RER Respiratory exchange ratio RPM Revolutions per minute TTE Time-to-exhaustion ?̇?O2max Maximal oxygen consumption VT Tidal volume WU Warm up xi Acknowledgements The following work would not have been possible without the strong support I have received throughout this project. First, I would like to thank Dr. Michael Koehle for agreeing to supervise me throughout my Master’s degree. Thank you for all your guidance and support throughout these past two years, with respect to not just this project but my own career and personal goals. I am glad you agreed to take “one more student,” and I look forward to the opportunity to work together in the future. Second, to my committee members Dr. Bill Sheel and Dr. Valérie Bougault, thank you for taking the time out of your own academic work to provide expertise for this project and support this journey. Thank you for all your feedback, insightful questions, and navigating time zone differences for meetings! Third, I would like to thank everyone who has worked out of Medical Sciences Block C or Chan Gunn Pavilion over the past two years; you have made this journey incredibly fulfilling and I am lucky to have worked with all of you. Thanks for providing data collection support, engaging in conversations, and making me happy to come into the lab every day. Fourth, thank you to all the athletes who volunteered their time, sweat, blood, and lungs for this study, with only minimal complaining… I would also like to acknowledge the financial support I have received from the Natural Sciences and Engineering Research Council of Canada, the Robert Caton & David Bates Scholarship Committee, and the UBC Faculty of Education. xii Dedication To my family, friends, and all those who have supported me through this endeavour. Chapter 1: Introduction 1.1 Background While ozone (O3) exists within the upper atmosphere (stratosphere) and protects humans from harmful incoming solar radiation, it also exists within the lower atmosphere (troposphere) as a pollutant (Mustafa, 1990). O3 within the troposphere is commonly referred to as ground-level O3, and is created by the interaction between nitrogen oxides, volatile organic compounds, and solar radiation, with production enhanced during periods of high temperatures (Gomes et al., 2010). The chemistry of O3 formation has been described in detail elsewhere (Mustafa, 1990). In brief, nitrogen dioxide (NO2) undergoes photolysis to produce atomic oxygen, which reacts with molecular oxygen to form O3 (Mustafa, 1990). Assuming a lack of competing reactions, a cyclic process occurs in which O3 reacts with NO to regenerate NO2 and O2, and O3 is unable to significantly rise until all available NO is converted to NO2, while secondary processes also exist in which chemical species create radicals to directly increase production of O3 (Mustafa, 1990). In urban areas, NOx is typically plentiful due to transportation and manufacturing emissions, so O3 formation is modulated by the presence of VOCs (Xiong & Du, 2020). Worldwide movement towards greater urbanization has increased population exposure to potentially hazardous pollutants (Baldasano et al., 2003). Of the numerous pollutants that exist within the atmosphere, ground-level ozone is the most established to have adverse effects on pulmonary function and development of negative respiratory symptoms (Hung et al., 2021). As O3 is a powerful oxidant, it interacts with the respiratory system both via direct reactions, and indirectly through free radicals and reactive intermediates (Mustafa, 1990). When inhaled, the interaction of O3 with the lining of the respiratory tract causes various negative effects depending on exposure length and concentration (Gomes et al., 2011). 2 While mechanisms are debated, O3 likely impacts the respiratory tract via two main pathways: the activation of sensory nerves in the respiratory tract, and respiratory tract injury, inflammation, and reactive oxidative stress (ISA for Ozone and Related Photochemical Oxidants, 2020). The stimulation of sensory nerves within the respiratory tract by O3 exposure activates the lung irritant response, causing bronchoconstriction of airways via autonomic and local responses to reduce ventilation and protect the lungs from potentially damaging chemicals (Kim et al., 2011). C-fiber endings and rapidly acting pulmonary receptors are largely responsible for this reflexive response (Lee & Widdicombe, 2001). The protective bronchoconstriction and irritant response are suggested to influence the ability to fully inflate the lungs and increase respiratory symptoms, which may result in shallower breathing in individuals exposed to O3 (Gibbons & Adams, 1984). Indeed, numerous studies have indicated a reduction in tidal volume with a corresponding increase in respiratory frequency with exposure to ozone (Folinsbee et al., 1977; Gibbons & Adams, 1984; Lauritzen & Adams, 1985; McDonnell et al., 1983). Additionally, the activation of irritant receptors likely negatively impacts initial flows during spirometry (McDonnell et al., 1983), as well as total lung capacity and vital capacity due to inability to fully inflate the lungs (Schelegle & Adams, 1986). Whether this response is voluntary in order to mediate increased respiratory symptoms, or an involuntary reflex response (Silverman et al., 1976), this less efficient method of breathing and corresponding symptoms may influence pulmonary function and therefore ventilation during exercise. Respiratory tract injury, inflammation, and oxidative stress directly damage the respiratory system, leading to increased airway responsiveness and airway obstruction (ISA for Ozone and Related Photochemical Oxidants, 2020). Inflammation is induced by reactive oxygen species (ROS) generation in response to ozone exposure, and can be measured via neutrophil 3 index of the pulmonary airways (Kim et al., 2011). Increased flow resistance and expiratory flow limitation due to obstruction of small to medium sized airways is likely a key factor related to the decrease in mid-expiratory flows during resting spirometry following O3 exposure (Silverman et al., 1976), as well as potential reductions in inspiratory reserve capacity due to gas trapping (Folinsbee et al., 1977). During exercise, it has been postulated that this effect may only be prevalent at maximal ventilation levels, with previous studies showing no change at anaerobic threshold (Savin & Adams, 1979). While correlations may be expected based on relationships between chronic inflammation and reduced pulmonary function in populations with respiratory diseases, such as asthma (Murdoch & Lloyd, 2010), or correlations between substance P immunoreactivity and fall in expiratory flows (Krishna et al., 1997), a lack of correlation exists between measured O3-induced inflammation and pulmonary function responses (Kleeberger, 1995). This indicates the complicated interplay of differing genetic mechanisms as to functional and cellular responses during O3 exposure (Kleeberger, 1995). Hence, lung function is decreased and respiratory symptoms may become more prevalent based on these two mechanisms during O3 exposure (ISA for Ozone and Related Photochemical Oxidants, 2020). A recent systematic review of randomized control trials indicated strong support for a reduction in pulmonary function following exercise as measured via spirometry with O3 exposure (Hung et al., 2021). Measures commonly employed in spirometry include forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), peak expiratory flow rate (PEFR), and forced expiratory flow from 25-75% of expiration (FEF25-75). Generally, decreases in FVC and FEV1 are found to be unimodal and negatively skewed in response to O3 exposure, indicating that while most participants have negative effects, the severity of such effects is varied (Kleeberger, 1995). It is likely that initial flow decreases during spirometry are 4 related to reduced inspiration based on activation of irritant receptors, whereas decreases throughout the rest of the trial are due to bronchoconstriction of smaller airways (McDonnell et al., 1983). FEF25-75, which is typically representative of flow through medium-to-smaller airways, is also reduced, while it does not follow the same unimodal pattern (Kleeberger, 1995). With exposure to greater concentrations of ozone, greater changes in pulmonary function and symptom development can be observed, indicating a dose-response relationship (Gong et al., 1986; Silverman et al., 1976). Some studies suggest a sigmoidal response, wherein the decrease in lung function plateaus above a certain ozone concentration level (McDonnell et al., 1983). However, pollutant exposure is best characterized by calculating the effective dose, based on the concentration of the pollutant in question, length of exposure, and the ventilation of the exposed individual (Adams & Schelegle, 1983). Hence, even studies administering low doses of ozone (60 ppb) can find consistent reductions in FVC and FEV1 if exposure duration is lengthened to over 6 hours (Kim et al., 2011). Furthermore, studies combining ozone exposure over a longer duration (i.e., up to 2 hours) with intermittent exercise, as opposed to simply rest, see larger decreases in lung function with increased minute ventilations (Silverman et al., 1976). Despite scientific consensus on the negative influence of O3 exposure on pulmonary function, variation exists when analyzing the exercise response across randomized control trials (Hung et al., 2021). While it seems logical that a reduction in pulmonary function in conjunction with negative respiratory symptoms will impair exercise performance, no agreement has been reached. Early studies utilizing high (750 ppb) doses of O3 demonstrated lower maximal oxygen consumption, heart rate, and workloads during a graded exercise test (Folinsbee et al., 1977), while others utilizing lower doses (300 ppb) saw only nonsignificant trends towards these variables, while showing significant decreases in maximal ventilation achieved in O3 conditions 5 (Savin & Adams, 1979). These studies indicate that reduced exercise capability may be linked to physiological ventilatory limitation, or to discomfort related to respiratory symptoms (Folinsbee et al., 1977). Well-trained athletes were more likely to complete competitive cycling simulations in lower concentrations of O3 or filtered air, whereas exposures up to 240 ppb O3 resulted in greater rate of dropouts (Schelegle & Adams, 1986). The authors also postulated that training status of participants may play an important role in likelihood of responses due to higher sustainable ventilation (Schelegle & Adams, 1986). Accordingly, elite athletes demonstrated significant decreases in ?̇?O2max, time-to-exhaustion, and minute ventilation during exposure to 200 ppb O3 during an incremental test following 60 minutes of constant work rate cycling, although no significant differences were found at 120 ppb (Gong et al., 1986). On the other hand, a multitude of studies have not demonstrated a difference in exercise capabilities during ozone exposure. Despite concentrations of ozone ranging between 250 ppb and 750 ppb, no differences in performance were noted during an incremental treadmill test to exhaustion (Horvath et al., 1979). Other studies also using running as the mode of exercise found no changes in an 8-kilometre time trial performance at 100 ppb O3, while differences were observed between high and low temperature trials (Gomes et al., 2010). When performing competitive cycling simulations under 240 ppb O3 conditions, no differences were seen for heart rate, breathing pattern, or oxygen consumption between conditions, despite differences in completion rates (Schelegle & Adams, 1986). While conflicting evidence exists, many studies note the wide range in individual responses to O3 exposure during exercise, limiting the ability to draw significant conclusions (Kleeberger, 1995). Inconsistency within scientific literature is largely attributed to interindividual differences in response to O3 exposure (Kleeberger, 1995), wherein a wide range of performance and 6 spirometry changes occur despite identical ozone dosage. Many studies recognize that individuality of responses to ozone in reference to pulmonary function (Gomes et al., 2010; McDonnell et al., 1983) and exercise response (Gomes et al., 2010; Schelegle & Adams, 1986), even within healthy participants, indicating that some individuals may be more prone to adverse responses to ozone exposure. In addition, both those with chronic respiratory diseases and female participants may be more likely to experience negative effects. Patients with increased airway hyperreactivity, such as asthmatics tend to respond more strongly than healthy individuals (Kleeberger, 1995). Further differences have been observed in airway hyperreactivity (Birukova et al., 2019) and breathing patterns (Lauritzen & Adams, 1985) during exercise between male and female participants, with females typically showing greater negative responses. Although mechanisms are not fully understood, variations in responsiveness between sexes may be related to smaller lung size (Gibbons & Adams, 1984); greater pollutant-to-lung ratio and alveolar surface area-to-lung ratio (Lauritzen & Adams, 1985), or other factors related to interindividual variability. Some studies suggest that while increased airway hyperresponsiveness in females is not related to progesterone levels, estrogen may have a protective effect on ozone-induced lung inflammation (Birukova et al., 2019). However, other studies show lower inflammatory values in female participants than males, despite greater decreases in pulmonary function in females (Kim et al., 2011). As such, the concept of “responders” and “non-responders” to O3 exposure has been postulated, as while certain characteristics such as sex, age, respiratory disease, or training status may increase likelihood of responses, even within homogenous groups of individuals, significant variability exists (Kleeberger, 1995) Previous literature has mainly focused on increasing the effective dose of O3 by subjecting participants to longer duration, low-intensity exercise in O3 conditions (Hung et al., 7 2021). While this may be suitable for understanding the chronic effects of O3 exposure (Lippmann, 1989), this is not typical of human exercise patterns. The Canadian Physical Activity Guidelines suggest that adults should complete a minimum of 150 minutes of moderate-to-vigorous physical activity per week (Tremblay et al., 2011). Thus, it is unwise to draw comprehensive conclusions from low-to-moderate intensity level exercise in O3 polluted environments. For example, during competition and high-intensity exercise, athletes inspire high volumes of ambient air, increasing the effective dose of pollution in a relatively short time period when compared to resting or low-intensity exercise (Hung et al., 2021). As athletes are typically only separated by small performance margins, particularly at the national and international level of competition, minor changes in performance can have large consequences on competition outcome (Sandford et al., 2021). With most athletic competitions, such as the Olympic Games, being held in or near metropolitan areas, the adverse effects of O3 are often prevalent due to higher pollutant concentrations (Peiser & Reilly, 2004). Exercise performance may be influenced by changes in pulmonary function that stem from the narrowing of small airways and dynamic hyperinflation of the lungs, which increases the metabolic cost of breathing (Silverman et al., 1976), the development of uncomfortable respiratory symptoms (Savin & Adams, 1979), inefficient ventilatory pattern (Folinsbee et al., 1975), or diffusion challenges (Young et al., 1964). A clear understanding of how O3 exposure impacts cardiorespiratory physiology during high-intensity exercise, and which athletes may be at greater risk is critical for developing strategies for predicting and mitigating its effects. 1.2 Research Question The purpose of this thesis was to assess the interaction between O3 exposure and cardiorespiratory function during rest and exercise in healthy athletes. We aimed to characterise 8 both resting and exercise pulmonary function response to O3, as well as to assess the effects of O3 exposure on ventilatory patterns and exercise parameters during submaximal and maximal exercise. 1.3 Hypothesis It was hypothesized that acute exposure to O3 would lead to decreases in pulmonary function during both rest and exercise, ventilatory pattern would be shifted towards more frequent and shallow breathing, and exercise capability during O3 exposure would be reduced when compared to room air. 9 Chapter 2: Methods 2.1 Sample and Recruitment The target population for this study was healthy male and female athletes with a competitive background in cycling or other similar endurance-based sports, such as running, swimming, or cross-country skiing. The sample representing this population was male and female individuals with a background in competitive endurance sports living and training in the Greater Vancouver area. Recruitment took place by non-probabilistic sampling methods, mainly by purposive sampling. Posters, which were written in lay language, and designed to outline the inclusion and exclusion criteria for partaking in the study, the purpose and study design, potential risk and benefits to participating, and contact information for the primary investigator were distributed via email and social media to local athletic clubs with athletes that may fit the inclusion and exclusion criteria to participate in the study (i.e., UBC Thunderbirds Cycling Sports Club, UBC Thunderbirds Triathlon Sports Club, Glotman-Simpson Cycling Club). Table 2.1 Inclusion and exclusion criteria. Inclusion Criteria Exclusion Criteria 1) Currently training and/or competing in endurance sport 2) Maximal oxygen consumption (?̇?O2max) greater than 60 ml/kg/min (males) or 50 ml/kg/min (females) 3) Between the ages of 18 and 50 4) Able to communicate sufficiently using the English language 5) Taking hormonal birth control (female participants only) 1) History of smoking 2) Upper respiratory tract infection within the last 4 weeks 3) Presence of any chronic respiratory disease 4) Current symptoms of, or current infection with COVID-19 5) Pregnant or suspected pregnancy Table 2.1 above outlines the inclusion and exclusion criteria for the research study. The first and second inclusion criterion: athletes currently training and/or competing in endurance sport, and maximal oxygen consumption (?̇?O2max) greater than 60ml/kg/min (males) or 10 50ml/kg/min (females) represents the target population for this study. Although elite endurance athletes have been shown to achieve ?̇?O2max values well above 70 ml/kg/min (Jones et al., 2021), such athletes were unlikely to participate in the proposed study to avoid interrupting their rigid training programs. The specified ?̇?O2max requirement allowed for well-trained individuals well above the 90th percentile of aerobic fitness (Kaminsky et al., 2022), without the time and training constraints of professional athletes to participate. Additionally, by reducing the variability of fitness level between participants, the differences in minute ventilation during exercise was also reduced between participants, helping ensure that a similar effective dose of pollution was delivered to all participants. The third inclusion criterion, between the ages of 18 and 50, reduced the likelihood of potential harm to participants from ozone exposure and exercise. The human respiratory tract is particularly vulnerable to air pollution exposure during periods of development, which may lead to short- and long-term health effects (Goldizen et al., 2016). As the respiratory tract is not fully developed until roughly 18-20 years old (Goldizen et al., 2016), only participants over the age of 18 were permitted to participate. Similarly, based on the increased risk of cardiovascular and respiratory diseases (Manisalidis et al., 2020), as well as cognitive impairment (Gao et al., 2022), adults over the age of 50 were not permitted to participate. The fourth inclusion criterion, ability to communicate sufficiently in the English language, ensured that participants were able to fully understand study procedures, ask questions, and provide informed consent. The fifth inclusion criteria, taking hormonal birth control (females only), minimized potential confounding factors on inflammatory responses in females due to the potential protective effects of estrogen (Birukova et al., 2019), and the stimulatory effects of progestin on ventilation (Brodeur et al., 1986). By removing variation in sex hormones that naturally occurs 11 throughout the menstrual cycle between experimental visits, greater confidence can be had in the results. The first and second exclusion criteria, history of smoking and presence of upper respiratory tract infection in the last 4 weeks, avoided potential biasing of spirometry and exercise measurements by ensuring a homogenous sample of healthy individuals, without changes in health status between experimental visits. The third exclusion criteria, presence of any chronic respiratory disease, helped mitigate the influence of such diseases on spirometry measures. Those with a diagnosis of respiratory diseases, such as asthma, have been shown to experience greater decreases in pulmonary function in response to ozone than healthy individuals (Kleeberger, 1995). As this study aimed to establish susceptibility and ventilatory responses to ozone in healthy participants, removal of participants with chronic respiratory diseases limited potentially conflicting factors. The fourth exclusion criteria, current symptoms of or infection with COVID-19, helped mitigate any potential influences of both short- and long-COVID-19 symptoms, as well as protected members of the research team. Symptoms commonly attributed to COVID-19 include difficulty breathing, coughing, and general weakness (Çalıca Utku et al., 2020) that may impact ability to properly perform exercise or spirometry measures. The fifth exclusion criteria, pregnant or suspected pregnancy, minimized risks to participants. As O3 exposure is shown to lead to negative health outcomes in pregnant women, such as increasing likelihood of preterm birth (Rappazzo et al., 2021), avoiding testing of participants who may have been pregnant increased participant safety. 12 2.2 Sample Size Calculation Sample size was calculated using data from previous studies that examined changes in FEV1 in athletes following O3 exposure during exercise. A similar crossover design in elite athletes utilizing ozone exposure at 200 ppb found a 0.95-litre reduction in FEV1 following exercise, with a standard deviation of post-exercise measures of 0.86-litres (Gong et al., 1986). Calculation for a crossover design based on analysis of variance F-tests using these values with a power of 0.8 and an alpha level of 0.05 led to a sample size of 15 participants. The decision to include both male and female participants resulted in an increase to 20 participants. 2.3 Measures/Instruments O3 was produced through the ACT-5000 Ozonetech corona discharge generator, (Mellifiq, Sweden), and mixed with room air in a sealed 3m x 3m x 2m chamber. O3 was delivered to participants via a tube connected to the inspiratory opening on a three-way non-rebreathe valve with a mouthpiece. The Thermo ScientificTM 49iQ Ozone Analyzer (Franklin, MA, USA) was attached to the inspiratory tube and used as an O3 sensor. Calibration occurred according to the manufacturing standards using the Thermo ScientificTM 49iQ Ozone Primary Standard generator. Spirometry was measured with the ParvoMedics OUS-SPIRO on the metabolic cart (Salt Lake City, UT, USA), which was calibrated prior to each use. While wearing a nose-clip, participants were asked to complete a maximal inspiration to maximal lung capacity at the end of a normal expiration, followed by a maximal forceful exhalation for a minimum of 6 seconds. This allowed for measurement of FVC, FEV1, FEV1/FVC, PEFR, and FEF25-75. Based on guidelines provided by the American Thoracic Society (ATS), trials were repeated a minimum of three times until the two largest FEV1 and FVC values differed by less than or equal to 150mL 13 (Graham et al., 2019). Spirometry values were compared between the two experimental trials pre- and post-exercise, as well as prior to and following the resting ozone exposure to examine how O3 exposure impacted pulmonary function. Symptoms were measured via a custom brief questionnaire previously used in the laboratory. Before and after each exercise bout and ozone exposure, participants were asked to rate symptoms related to their eyes, nose, throat, chest, and general health on a 0-5 scale, where 0 means “you don’t experience this symptom at all” and 5 means “very severe”. Symptoms were compared between the two experimental trials pre- and post-exercise, as well as prior to and following the resting ozone exposure to examine how O3 exposure impacted the development of certain symptoms. Dyspnea during rest and exercise was measured using a modified Borg scale (Gaber et al., 2019). During rest and exercise, participants will be asked “How strong/intense is your breathing discomfort?” in which zero will represent “no breathing discomfort” and 10 represents “the most severe breathing discomfort that you have ever experienced or could imagine experiencing.” Ratings of dyspnea were compared between the two experimental trials pre- and post-exercise, as well as prior to and following the resting ozone exposure to examine how O3 exposure impacted feelings of breathlessness in the lungs. Rating of perceived exertion (RPE) during exercise was measured using the Borg scale (Borg, 1982). Participants were intermittently asked to express how hard they felt like they were working, on a scale from 6 to 20, where a 6 represents “no exertion” and a 20 represents “maximal exertion.” Ratings of perceived exertion will be compared between the two experimental trials to examine how O3 exposure may impact RPE, as well as assess each subject’s state during the graded exercise test. 14 Blood lactate was measured via testing strips and a portable LactatEDGE analyzer (Warsaw, Poland) by sampling at the fingertip of the ring or middle finger. The skin surface was prepared by cleaning with an alcohol swab, wiping with gauze, followed by puncturing the skin with a lancet. After wiping the first drop of blood, the second drop was analyzed for concentration of blood lactate (mmol/L). The discarded lancet was placed in a designated sharps bin, while any other materials exposed to blood (gauze, alcohol swab, discarded strips) were disposed of in the marked biohazard bin. The primary researcher wearing gloves obtained all samples. Blood lactate values were utilized to determine aerobic and anaerobic thresholds during the step-incremental cycling test to prescribe individualized work rates for experimental trials, as well as to ensure that prescription of work rates placed participants within the correct training zones. Blood lactate data from the step-incremental exercise test was 3rd degree polynomial fitted and graphed against power output for determination of first and second lactate thresholds through an online app designed to simplify this process (Keir et al., 2022). While various models and strategies exist with minimal consensus within scientific literature as to their applicability, the first and second lactate thresholds were determined based on the “Log-Log” method and onset of blood lactate (OBLA) of 3.5mmol/L, respectively. The Log-Log method is commonly used to determine the first systematic increase in blood lactate values in high level athletes, representing the transition between moderate and heavy exercise intensity in the classic 3-zone model (Rogers et al., 2022). By removing visual selection, greater resolution can be had as to the transition point in blood lactate values (Beaver et al., 1985). Anaerobic threshold was determined as the work rate associated with blood lactate at 3.5mmol/L. Review articles demonstrate strong correlations between onset of blood lactate (OBLA) of 4mmol/L established during step- 15 incremental exercise tests and maximal lactate steady state (MLSS), although generally overestimating sustainable work rate based on data from 3-minute stages (Faude et al., 2009). To rectify this potential issue, a more conservative OBLA value of 3.5mmol/L was utilized for this study. This strategy has been previously used in studies examining endurance-trained males with significant correlations between OBLA 3.5mmol/L and MLSS (Denadai et al., 2004). Inspiratory capacity maneuvers were performed during rest and exercise to assess ventilatory patterns. While connected to the mouthpiece, participants were asked to fill their lungs completely from the end of a normal expiration. The volume of air inspired during these tasks represented the inspiratory capacity of each subject, and was compared between the two experimental trials to examine how O3 exposure impacted dynamic hyperinflation of the lungs during exercise. Individualized work rate for the verification exercise bout (Visit 1) and time-to-exhaustion trials (Visit 3 & Visit 4) was determined based on the performance during the step-incremental exercise test based on the following formula: Maximal Power = Last Completed Stage (W) + (Time of Last Stage/180 x 25) “Maximal Power” and “Last Completed Stage” was represented in watts (W), and “Time of Last Stage” in seconds. The resulting Maximal Power was then multiplied by 1.05 to determine the work rate associated with 105% of Maximal Power. This formula allowed for partial credit for incomplete stages, which was common during the step-incremental protocol. 16 2.4 Study Design This research design can be characterized as a double-blind crossover with pre- and post- measures. Ozone pollution status was the independent variable, of which there were two levels during the experimental trials: 170 ppb O3, and room air (<10 ppb O3). Testing took place over four laboratory sessions, with a minimum of 72 hours of washout between each visit. During the first session, participants completed a step-incremental test on a cycle ergometer to determine participant characteristics and identify lactate thresholds, familiarization of exercise pulmonary function tests, and a familiarization time-to-exhaustion (TTE) test. During the second visit, participants completed resting spirometry before and after exposure to 120 minutes of O3 at 750 ppb to characterize resting response. The third and fourth laboratory visits consisted of 25 minutes of cycling at the workload associated with 10% below aerobic threshold (warmup) and 30 minutes of cycling at the workload associated with 10% below previously established anaerobic threshold (data collection). Post-exercise spirometry was obtained prior to and following a TTE test at 105% of previously established maximal power output. During the O3 condition, participants were exposed to 170 ppb throughout the cycling tasks, whereas during the control condition participants breathed room air for the identical procedures. Both the primary researcher and participants were blinded to the experimental condition for each visit, with the order randomized for each participant and counterbalanced so the same number of participants experienced each order of conditions. 2.5 Procedures Participants visited the laboratory for testing on four occasions. A minimum of 72 hours of washout was required between visits. Before all visits, participants were asked to avoid 17 exercise for a minimum of 6 hours to minimize bronchodilation of airways prior to resting spirometry values (Gotshall, 2006). Before each session involving exercise (Visits 1,3,4), participants were asked to prepare as if each visit was a competition or important training session in their training schedule, and to ensure preparation is as similar as possible prior to each visit to minimize variability in results. This included, but was not limited to, avoiding alcohol and strenuous exercise for 24 hours, ensuring a proper sleep the night prior, and consuming a pre-exercise meal to which they were accustomed, timed appropriately prior to each visit. However, participants were advised not to consume any ergogenic aids (i.e., sodium bicarbonate) or stimulants (i.e., significant doses of caffeine) for 24 hours prior to each visit, even if they typically utilized such strategies during competition. Experimental visits (3,4) were performed at a similar time of day in order to avoid any diurnal variation in exercise performance (Kusumoto et al., 2021). The first visit involved participant consent, screening for inclusion based on a maximal exercise test, familiarization with pulmonary function measurements, and a familiarization TTE test. Participants first re-read the consent form, and were given the opportunity to ask any questions related to their involvement, before being asked to sign the informed consent. Participants also completed a physical activity readiness questionnaire (PAR-Q+) (Warburton et al., 2011), which was reviewed prior to partaking any exercise in the laboratory. Participants were then familiarized with pulmonary function testing equipment and protocols, and underwent resting spirometry. Spirometry involved maximal inspiration followed by maximal forceful expiration to determine FEV1, FVC, FEV1/FVC, FEF25-75, & PEFR. Following resting pulmonary function measurements, participants adjusted the Velotron DynaFit Pro cycle ergometer (Racermate Inc., Seattle, U.S.A.) to a comfortable position, which was recorded to 18 ensure that the position remained identical between visits. Participants then completed a 10-minute warmup at 1 watt/kilogram body weight, before performing a step-incremental graded exercise test (GXT). Participants were fitted with a mouthpiece connected to a three-way non-rebreathe valve, and expired gas and flow were analyzed by a ParvoMedics TrueOne 2400 metabolic measurement system (Sandy, UT, USA) for all exercise tests. The step-incremental GXT consisted of 3-minute stages, starting at 100 watts (males) or 75 watts (females) with power output increasing by 25 watts/stage, until the participant reached volitional exhaustion, was unable to maintain 60 revolutions per minute (RPM), or was unwilling to continue. Blood lactate was sampled via finger prick during the last 10 seconds of each stage. Ratings of perceived exertion was also collected at the end of each stage. Following the completion of the test, participants were given a brief recovery before cycling for a maximum of 5 minutes at the power output associated with 10% below their anaerobic threshold to mimic experimental visits 3 and 4. During this constant workload bout, participants were instructed as to how to complete an inspiratory capacity (IC) maneuver, as well as provide rating of perceived exertion and dyspnea. Participants then performed post-exercise pulmonary function measures identical to that completed at rest, prior to completing a TTE test at 105% of the previously achieved maximum work rate. Participants were asked to cycle for as long as possible at the specified work rate until they were no longer able to maintain 60 rpm, and time-to-exhaustion was recorded. Participants were blinded as to time completed throughout the trial, and were only able to view their cadence to ensure that their rpm did not drop below 60. Participants then performed a final round of pulmonary function measures. The second visit consisted of resting exposure to O3 and pulmonary function tests. Participants completed pre-O3 resting spirometry, dyspnea, and symptom measurements, 19 followed by 2 hours of resting O3 exposure at 750 ppb. During the resting exposure, participants were seated with a three-way non-rebreathe valve and mouthpiece connected via tube to the ozone chamber. Nose-clips were worn for the entirety of the protocol to ensure only oral ventilation was utilized. Participants were permitted to use personal electronic devices (phone, laptop, tablet etc.) during exposure, provided no significant movement was required. Following exposure, participants completed identical spirometry, symptom, and dyspnea measurements immediately and 30 minutes post-exposure to that completed pre-exposure. Post-exposure and pre-exposure values were compared to determine resting response to O3. The third and fourth visits consisted of identical procedures, randomized as to the order of the O3 and control conditions. During both experimental trials, a lab member other than the primary researcher controlled the O3 concentration in order to maintain double-blinding of the participant and the researcher to the condition. During the O3 exposure, participants received 170 ppb O3, and during the control exposure participants received room air (typically <10 ppb O3). Upon arrival in the laboratory, the primary researcher confirmed that pre-visit requirements (detailed above) were met, prior to resting spirometry, symptoms, blood lactate, and dyspnea measurements. Participants were then connected via tube and mouthpiece/valve to the O3 chamber. While remaining connected to the O3 chamber, participants completed an individualized warmup on the Velotron of 25 minutes at 10% below previously established aerobic threshold. Participants were then provided with 5 minutes of rest, during which a post-warmup blood lactate measurement was obtained, prior to completing 30 minutes at 10% below anaerobic threshold. All submaximal exercise intensities were rounded to the nearest 5-watt increments. Expired gas and volumes were analyzed throughout the trial, and participants performed an inspiratory capacity test, had blood lactate sampled, rated their perceived exertion 20 and dyspnea every 10 minutes (10, 20, and 30-minutes into exercise). Participants were encouraged to maintain a steady, comfortable cadence, and to match cadence between experimental visits. Post-exercise, participants removed their mouthpiece and completed spirometry, dyspnea, and symptom testing (~15mins), prior to reattaching the mouthpiece and completing a time-to-exhaustion test at 105% of peak power achieved during the GXT. Strong verbal encouragement was provided to participants, and exercise was stopped when they were no longer able to maintain 60 RPM, and time-to-exhaustion was recorded. Post-exercise, a final set of pulmonary function, symptom, and dyspnea testing occurred (~30 minutes following completion of the submaximal exercise bout). Figure 2.1. Example of instrumentation during experimental visits. Participants were seated on the Velotron cycle ergometer, with nose clips and a three-way non-rebreathe mouthpiece. Expired gas and volumes were measured at the metabolic cart (A), whereas the experimental air condition was delivered through the inspiratory tube (B) from the pollution chamber (C). 21 Figure 2.2. Ozone concentration control setup. Ozone was generated by the Ozonetech ACT5000 generator, and operated by a second researcher behind a curtain to maintain double-blinding. Concentration was adjusted via a dial (A), and real-time concentrations were viewed on the screen (B). 2.6 Data Analysis As the purpose of this research study was to assess the interaction between O3 exposure and cardiorespiratory function during rest and exercise in healthy athletes, we aimed to characterise the relationship between resting response and exercise response, as well as to assess the effects of O3 exposure on pulmonary function, ventilatory patterns and exercise parameters. All data analysis was performed with Statistical Package for Social Science (SPSS). 22 Chapter 3: Results 3.1 Participant Recruitment A total of 84 potential participants (Males, n=39, Females, n=45) contacted the research group regarding the study. After details of the study were provided via email, 26 did not follow up further. Others were deemed ineligible over email by the research staff based on not using hormonal contraceptives (n=11), physical fitness (n=6), missing the recruitment window (n=12), medical contraindications (n=2), age (n=1), or did not disclose a specific reason for not wishing to complete the study (n=1). Of the 24 participants who completed a screening visit (Males, n=13, Females: n=11), 20 completed all four study visits, with one dropout after one visit, and one dropout after two visits. Further participants were excluded based on not meeting the minimum ?̇?O2max requirement (n=1), and due to previously undisclosed medical contraindications (n=1). 3.2 Participant Characteristics Only participants who completed all study visits were included in subsequent analysis. Males (n=13) were 28 ± 7 years old, 181.8cm tall, and weighed 76.2 ± 6.5kg. Females (n=7) were 27 ± 5 years old, 167.2cm tall, and weighed 61.6 ± 5.3kg. The greatest 30-second ?̇?O2peak value achieved on any experimental day was 67.6 ± 5.7 ml/kg/min for males, and 57.6 ± 3.47 ml/kg/min for females. Primary sport of competition at the time of the study was cycling (n=14), followed by running (n=3), triathlon (n=2), and cross-country skiing (n=1). All participants were familiar with training and/or competing in cycling. First lactate threshold, based on the log-log method, was calculated as 208 ± 37 watts (2.61 ± 0.50 w/kg) for males, and 145 ± 18 watts (2.36 ± 0.36 w/kg) for females. 2nd lactate threshold, calculated based on the OBLA 3.5 method, was calculated as 274 ± 30 watts (3.61 ± 23 0.43 w/kg) for males, and 186 ± 21 watts (3.04 ± 0.44 w/kg) for females. Maximum power achieved during the ?̇?O2max test, with partial credit for incomplete stages, was 362 ± 29 watts (4.77 ± 0.45 w/kg) for males, and 260 ± 11 watts (4.24 ± 0.30 w/kg) for females. When comparing to normative values, males and females were of similar fitness and cycling ability. 3.3 O3 Concentrations During the resting O3 exposure, average O3 concentration was 750.3 ppb, while the maximum and minimum O3 concentrations for any participant were 752.9 ppb and 747.4 ppb, respectively. For the experimental visits, average O3 concentration for the O3 condition was 171.9 ppb, and 5.9 ppb for the room air condition. The highest O3 concentration for any participant in the O3 condition was 177.3 ppb, and the lowest was 167.9 ppb, whereas the maximum and minimum O3 concentrations in the room air condition were 15.0 ppb, and -0.2 ppb, respectively. All O3 concentrations were well matched to their target concentrations. 3.4 Resting O3 Pulmonary Function and Symptom Responses Repeated-measures ANOVAs were performed for FVC, FEV1, FEV1/FVC, PEFR, and FEF25-75 to compare changes in spirometry measures immediately and 30 mins post 2-hour exposure to 750 ppb O3. A main effect of time was present for FVC (F(1.267, 24.072) = 18.040, ηp2 = 0.487, p<0.001). FVC was lowest (5.17 ± 1.18L) 0 mins post-exposure compared to both baseline (5.69 ± 0.98L, p<0.001) and 30 mins post (5.36 ± 1.08L, p=0.030). FVC at 30 mins post remained significantly lower than baseline (p<0.001). A main effect of time was also found for FEV1 (F(1.242,23.600) = 30.509, ηp2 = 0.616, p<0.001). FEV1 was significantly decreased from baseline (4.52 ± 0.61L) at both 0 mins (3.89 ± 0.80L, p<0.001) and 30 mins post (4.10 ± 0.71L, p<0.001). FEV1 increased between 0 mins and 30 mins post (p=0.009). 24 FEV1/FVC also demonstrated a main effect of time (F(2,38) = 9.353, ηp2 = 0.330, p<0.001). Both 0 mins (78.0 ± 5.9%, p=0.007) and 30 mins post (78.3 ± 6.7%, p<0.001) were lower than baseline measurement (80.7 ± 6.0%). No differences were observed between either post exposure measures (p=1.000). A main effect of time was also shown for PEFR (F(1.360,25.842) = 30.389, ηp2 = 0.615, p<0.001). Post hoc tests showed that both 0 mins (8.09 ± 1.79L/s, p<0.001) and 30 mins post (8.80 ± 1.50L/s, p<0.001) were lower than baseline (10.19 ± 2.03L/s), with 0 mins also lower than 30 mins post (p=0.006). FEF25-75 also demonstrated a significant main effect of time (F(2,38) = 17.176, ηp2 = 0.475, p<0.001). 0 mins (3.53 ± 0.88L/s) and 30 mins (3.51 ± 1.20L/s) post-exposure were not different from each other (p=1.000), but were both lower than baseline (4.45 ± 0.96L/s, p<0.001). Development of symptoms was also analyzed using repeated-measures ANOVAs across baseline, 0 mins and 30 mins post 2 hours of exposure to 750 ppb O3. No main effects of time were observed for “watering eyes” (F(1.275,24.221) = 1.045, ηp2 = 0.052, p=0.336), “painful or stinging sensation in the eyes” (F(12,38) = 1.879, ηp2 = 0.090, p=0.167), “itchy nose” (F(1.260,23.935) = 3.524, ηp2 = 0.156, p=0.064), “runny nose” (F(1.516,28.802) = 3.585, ηp2 = 0.159, p=0.052), “painful or stinging sensation in the nose (F(1.012, 19.221) = 2.685, ηp2 = 0.124, p=0.117), “sneezing” (F(1.228,23.339) = 0.454, ηp2 = 0.023, p=0.547), “wheezing or whistling sounds in the chest” (F(1.298,24.656) = 3.123, ηp2 = 0.141, p=0.080), and “nausea” (F(1.403,26.662) = 1.662, ηp2 = 0.080, p=0.211). A main effect of time was found for “itchy eyes” (F(1.417,26.917) = 6.209, ηp2 = 0.246, p=0.012), but only differed between 0 mins (0.4 ± 0.6) and 30 mins post (0.1 ± 0.3, p=0.030)), with no differences between baseline (0.2 ± 0.5) and either post- measure (p=0.071, p=1.000, 25 respectively) based on Bonferroni corrected post hoc tests. A main effect of time was demonstrated for “blocked nose” (F(1.493,28.372) = 5.089, ηp2 = 0.211, p=0.020). 30 mins post (0.5 ± 0.7) was lower than both 0 mins post (1.3 ± 1.5, p=0.036) and baseline (1.0 ± 0.9, p=0.050), although baseline and 0 mins post were not different from each other (p=0.604). “Headache” also demonstrated a main effect of time (F(1.361,25.857) = 4.805, ηp2 = 0.202, p=0.028), but did not differ significantly between any time points (p>0.073). A main effect of time on “fatigue” (F(1.414,26.874) = 3.913, ηp2 = 0.171, p=0.045) was also shown, but only differed significantly between 0 mins (1.3 ± 1.2) and 30 mins post (0.8 ± 1.0, p=0.006), but not between baseline (0.8 ± 0.9) and either other time point (p=0.164, p=1.000, respectively). “Shortness of breath” showed significant main effect of time (F(1.417, 26.917) = 18.212, ηp2 = 0.489, p<0.001). Severity was greatest 0 mins post (2.1 ± 1.4) compared to both baseline (0.3 ± 0.7, p<0.001) and 30 mins post (1.1 ± 0.8, p<0.001), and remained higher at 30 mins post than baseline (p=0.030). “Dyspnea” also demonstrated a main effect of time (F(1.367,25.972) = 27.420, ηp2 = 0.591, p<0.001). Dyspnea was highest at 0 mins post (2.8 ± 2.2) compared to both baseline (0.2 ± 0.5, p<0.001) and 30 mins post (1.6 ± 1.6, p<0.001), while 30 mins post was still greater than baseline measurement (p=0.001). A main effect of time was shown for “chest tightness” (F(1.284,24.398) = 22.258, ηp2 = 0.539, p<0.001). Chest tightness was greater at both 0 mins (2.1 ± 1.6, p<0.001) and 30 mins post (1.2 ± 1.0, p=0.003) than baseline (0.3 ± 0.6), although 0 mins was greater than 30 mins post (p<0.001). “Chest pain” demonstrated a similar main effect of time (F(1.965, 37.326) = 6.423, ηp2 = 0.253, p=0.004). Chest pain was highest 0 mins post (0.9 ± 1.2) compared to baseline (0.1 ± 0.2, p=0.009), but no differences were observed at 30 mins post (0.5 ± 1.0, p=0.217), or between baseline and 30 mins post (p=0.264). A main effect of time was also observed for “dry, scratchy, 26 or sore throat” (F(1.317,25.031) = 5.316, ηp2 = 0.219, p=0.022), and was highest at 0 mins post (1.2 ± 1.2) compared to both baseline (0.3 ± 0.8, p=0.039) and 30 mins post (0.6 ± 1.2, p=0.003), with no differences between baseline and 30 mins post (p=1.000). Figure 3.1. Change in FVC (L) and FEV1 (L) following a 2-hour exposure to 750 ppb O3. * represents significantly different from baseline (p<0.05), and ** represents significantly different from all other time points (p<0.05). 33.544.555.566.577.58Baseline 0-min 30-minFVC (L)Time** **22.533.544.555.5Baseline 0-min 30-minFEV1 (L)Time** ** 27 Figure 3.2. Change in FEV1/FVC (%), PEFR (L/s), and FEF25-75 (L/s) following a 2-hour exposure to 750 ppb O3. * represents significantly different from baseline (p<0.05), and ** represents significantly different from all other time points (p<0.05). 65707580859095Baseline 0-min 30-minFEV1/FVC (%)Time* * 3.55.57.59.511.513.5Baseline 0-min 30-minPEFR (L/s)Time** **1.52.53.54.55.56.5Baseline 0-min 30-minFEF 25-75 (L/s)Time* * 28 3.5 Exercise Intensity Individualized exercise intensities for the WU and CWR cycling bouts were prescribed based on 90% of 1st and 2nd lactate thresholds, respectively. WU power output was 167 ± 40 watts for males (2.47 ± 0.47 watts/kg), and 130 ± 16 watts for females (2.13 ± 0.32 watts/kg). CWR power output was 247 ± 27 watts for males (3.25 ± 0.38 watts/kg), and 167 ± 19 watts for females (2.74 ± 0.40 watts/kg). Overall averages for power output were 186 ± 44 watts for WU (2.35 ± 0.45 watts/kg), and 219 ± 46 watts for CWR (3.07 ± 0.45 watts/kg). A repeated-measures ANOVA was used to confirm and compare exercise intensity for all submaximal cycling bouts between conditions. No significant condition-by-time interaction (F(2.572,48.873) = 2.190, ηp2 = 0.103, p=0.103) or main effect of condition (F(1.000,19.000) = 3.151, ηp2 = 0.142, p=0.092) was observed for blood lactate, although a main effect of time (F(1.780,33.819) = 32.241, ηp2 = 0.629, p<0.001) was observed. Blood lactate was not different between baseline (1.5 ± 0.4 mmol/L) and post-WU (1.5 ± 0.6 mmol/L), but both these times differed from all other points (p<0.001). Blood lactate was not different between 10 mins (2.6 ± 0.7 mmol/L), 20 mins (2.7 ± 0.8 mmol/L), and 30 mins (3.0 ± 1.2 mmol/L) into CWR-bout exercise (p>0.081). No significant condition-by-time interaction (F(2,38) = 0.617, ηp2 = 0.031, p=0.545) or main effect of condition (F(1,19) = 0.005, ηp2 = 0.000, p=0.947) was observed for RPE, while a main effect of time was demonstrated (F(1.370,26.030) = 62.655, ηp2 = 0.767, p<0.001). RPE increased significantly between all time points when measured at 10 mins (11.7 ± 1.3), 20 mins (12.8 ± 1.6), and 30 mins (13.3 ± 1.8) into CWR-bout exercise (p<0.001). TTE exercise intensity was individualized to 105% of the peak power achieved during the step-incremental ?̇?O2max test on the first study visit, taking into consideration both incomplete and complete stages. Power output for the TTE was 380 ± 30 watts for males (5.01 ± 29 0.47 watts/kg), and 273 ± 11 watts for females (4.45 ± 0.31 watts/kg). Overall average power output for the TTE was 342 ± 58 watts (4.81 ± 0.50 watts/kg). 3.6 25-min Warmup Exercise Response Two-tailed, paired-samples T-tests were utilized to compare metabolic data between O3 and room air conditions for the 25-min warmup period, with data presented as an overall average ± standard deviation. Relative ?̇?O2 did not differ between O3 (33.84 ± 5.99 ml/kg/min) and room air (33.65 ± 5.85 ml/kg/min) conditions (t = 0.567, ηp2 = 0.018, p=0.578). VCO2 was also not different between O3 (2065.22 ± 467.80 ml/min) and room air (2033.37 ± 459.45 ml/min) conditions (t = 1.288, ηp2 = 0.084, p=0.214). No change was observed for RER between O3 (0.861 ± 0.023) and room air conditions (0.854 ± 0.034) (t = 1.207, ηp2 = 0.075, p=0.243). Breathing frequency was also not different between O3 (25.52 ± 4.63 breaths/min) and room air conditions (25.51 ± 4.63 breaths/min, t = 0.015, ηp2 = 0.000, p=0.988). There was no difference between O3 (2.18 ± 0.50 L/breath) and room air (2.14 ± 0.40 L/breath) conditions for tidal volume (t = 0.015, ηp2 = 0.029, p=0.475). Minute ventilation also showed no change between O3 (54.27 ± 12.50 L/min) and room air (53.73 ± 12.50 L/min) conditions (t = 0.767, ηp2 = 0.032, p=0.453). ?̇?E/?̇?O2 was not different between O3 (22.74 ± 1.92) and room air (22.70 ± 1.99) conditions (t = 0.175, ηp2 = 0.002, p=0.863). There was no change in ?̇?E/VCO2 between O3 (26.37 ± 1.69) and room air (26.52 ± 1.50, t = -0.662, ηp2 = 0.024, p=0.516). Heart rate was also not different between O3 (134.4 ± 12.3 bpm) and room air (133.8 ± 11.8 bpm) conditions (t = 0.252, ηp2 = 0.004, p=0.804). 30 Table 3.1. Changes in metabolic variables during a 25 min warmup cycling bout at 90% of first lactate threshold. 170ppb O3 Room Air P-Value Relative V̇O2 (ml/kg/min) 33.84 ± 5.99 33.65 ± 5.85 0.578 VCO2 (ml/min) 2065.22 ± 467.80 2033.37 ± 459.45 0.214 RER 0.861 ± 0.023 0.854 ± 0.034 0.243 FB (Breaths/min) 25.52 ± 4.63 25.51 ± 4.63 0.988 VT (L/breath) 2.18 ± 0.50 2.14 ± 0.40 0.475 V̇E (L/min) 54.27 ± 12.50 53.73 ± 12.50 0.453 V̇E/V̇O2 22.74 ± 1.92 22.70 ± 1.99 0.863 V̇E/VCO2 26.37 ± 1.69 26.52 ± 1.50 0.516 HR (BPM) 134.4 ± 12.3 133.8 ± 11.8 0.804 3.7 30-min Constant Work Rate Bout Exercise Response Repeated measures ANOVAs were used to analyze potential differences over time and between conditions for metabolic data throughout the 30-min CWR exercise bout. Results are represented as 5-minute averages (0-5mins, 5-10mins, 10-15mins, 15-20mins, 20-25mins, 25-30mins) between conditions. Relative ?̇?O2 demonstrated no significant condition-by-time interaction (F(2.185,41.522) = 1.529, ηp2 = 0.067, p=0.269), or main effect of condition (F(1,19) = 2.602, ηp2 = 0.120, p=0.123). A main effect of time (F(2.668,50.693) = 261.024, ηp2 = 0.932, p<0.001) was demonstrated for relative ?̇?O2. Relative ?̇?O2 as not different between 10-15mins (43.38 ± 8.24ml/kg/min), 15-20mins (43.20 ± 8.24ml/kg/min), and 20-25mins (43.535 ± 8.06ml/kg/min) segments (p>0.878), while 0-5mins (39.026 ± 7.46) and 5-10mins (42.719 ± 31 8.10ml/kg/min) were different from all other time points (p<0.001). 25-30mins relative ?̇?O2 (43.65 ± 8.33ml/kg/min) was also different from 15-20mins (p<0.001). VCO2 did not demonstrate a significant condition-by-time interaction (F(2.447,47.064) = 0.770, ηp2 = 0.039, p=0.494), or main effect of condition (F(1,19) = 0.213, ηp2 = 0.011, p=0.649), while a main effect of time was present (F(2.224,42.247) = 176.645, ηp2 = 0.903, p<0.001). 0-5mins VCO2 (2382.87 ± 676.99 ml/min) was different from all other time points (p<0.001), and 5-10mins (2683.58 ± 750.98 ml/min) was different from all time points other than 15-20mins (2716.94 ± 776.61 ml/min, p=0.093). 10-15mins (2737.93 ± 779.41 ml/min) was not different than 15-20 (p=0.363), 20-25 (2744.74 ± 776.38 ml/min, p=1.000), or 25-30 mins (2744.83 ± 791.34 ml/min, p=1.000). No significant condition-by-time interaction was observed for RER (F(2.346,44.573) = 0.205, ηp2 = 0.011, p=0.848), while main effects of both condition (F(1,19) = 5.470, ηp2 = 0.224, p=0.030) and time (F(2.606,49.518) = 36.714, ηp2 = 0.659, p<0.001) were present. RER was greater in the O3 condition (0.884 ± 0.055) than room air (0.874 ± 0.066). 0-5mins RER (0.859 ± 0.038) was different than all other time points (p<0.001), while all other time points did not differ from each other (p>0.548). Breathing frequency did not demonstrate a significant condition-by-time interaction (F(1.621,30.803) = 2.035, ηp2 = 0.097, p=0.155), or main effect of condition (F(1,19) = 1.476, ηp2 = 0.072, p=0.239), while a main effect of time was present (F(1.587,30.162) = 86.353, ηp2 = 0.820, p<0.001). Frequency of breathing increased throughout the exercise bout, and was significantly different between adjacent time points (p<0.026) other than between 10-15mins and 15-20mins (p=0.153). 32 No significant condition-by-time interaction (F(1.433,27.236) = 2.156, ηp2 = 0.102, p=0.147), or main effect of condition (F(1,19) = 1.240, ηp2 = 0.061, p=0.279) was observed for tidal volume. A main effect of time was present (F(1.669,31.714) = 14.657, ηp2 = 0.435, p<0.001). Tidal volume increased between 0-5mins (2.334 ± 0.72 L/breath) and 5-10 mins (2.421 ± 0.71 L/breath), before decreasing throughout the remainder of the exercise bout, albeit only significantly between adjacent time points of 5-10mins and 10-15mins (p<0.001), and 10-15mins and 15-20mins (p=0.012). Minute ventilation did not demonstrate a significant condition-by-time interaction (F(2.153,40.904) = 5.814, ηp2 = 0.025, p=0.632), or main effect of condition (F(1,19) = 0.170, ηp2 = 0.009, p=0.684), while a main effect of time was present (F(1.8127,34.432) = 138.086, ηp2 = 0.879, p<0.001). ?̇?E generally increased with exercise duration, but not significantly between adjacent time points of 10-15mins and 15-20mins (p=1.000), and 20-25mins and 25-30mins (p=1.000). No significant condition-by-time interaction (F(2.000,38.007) = 0.651, ηp2 = 0.033, p=0.527), or main effect of condition (F(1,19) = 1.110, ηp2 = 0.055, p=0.305) was observed for ?̇?E/?̇?O2, while a main effect of time was present (F(2.024,38.462) = 39.333, ηp2 = 0.674, p<0.001). ?̇?E/?̇?O2 generally increased with exercise duration, but only significantly between adjacent time points of 0-5mins and 5-10mins (p<0.001), and 5-10mins and 10-15mins (p=0.002). ?̇?E/VCO2 did not show a significant condition-by-time interaction (F(1.028,20.448) = 0.955, ηp2 = 0.048, p=0.392) or main effect of condition (F(1,19) = 0.001, ηp2 = 0.000, p=0.978). A main effect of time was observed (F(1.908,36.253) = 37.551, ηp2 = 0.664, p<0.001), as ?̇?E/VCO2 generally increased with exercise duration after no change between 0-5mins and 5-10mins (p=1.000), although only significantly between adjacent time points of 5-10mins and 10-15mins (p<0.001), 15-20mins and 20-25mins (p=0.004), and 20-25mins and 25-30mins (p=0.005). 33 Heart rate demonstrated no significant condition-by-time interaction (F(1.611,30.611) = 1.260, ηp2 = 0.062, p=0.291) or main effect of condition (F(1,19) = 0.004, ηp2 = 0.000, p=0.949), although a main effect of time was found (F(1.645,31.251) = 83.287, ηp2 = 0.814, p<0.001). HR generally increased as exercise duration continued, although insignificantly between adjacent time points of 10-15mins and 15-20mins (p=0.355), and 20-25mins and 25-30mins (p=1.000). A significant condition-by-time interaction was observed for inspiratory capacity (F(2,36) = 4.942, ηp2 = 0.215, p=0.013), although post-hoc tests did not demonstrate significant differences between conditions at any time points (p>0.055). In the O3 condition, 20-min IC was greater than 30-min IC (p=0.035), and in the RA condition, 10-min IC was greater than 20-min IC (p=0.011). No condition (F(1,18) = 0.565, ηp2 = 0.030, p=0.462) or time (F(1.504,27.078) = 0.132, ηp2 = 0.007, p=0.818) main effects were present. Figure 3.3. Change in RER throughout 30-minute constant work-rate bout at 90% of LT2. * represents significantly different between conditions at a given time point (p<0.05), and ** represents a main effect of condition (p<0.05). 0.750.770.790.810.830.850.870.890.910.930.950-5min 5-10min 10-15min 15-20min 20-25min 25-30minRespiratory Exchange RatioTimeO3RA** 34 Figure 3.4. Change in breathing frequency throughout a 30-minute constant work-rate bout at 90% of LT2. * represents significantly different between conditions at a given time point (p<0.05), and ** represents a main effect of condition (p<0.05). Figure 3.5. Change in tidal volume throughout a 30-minute constant work-rate bout at 90% of LT2. * represents significantly different between conditions at a given time point (p<0.05), and ** represents a main effect of condition (p<0.05). 1520253035404550550-5min 5-10min 10-15min 15-20min 20-25min 25-30minBreathing Frequency (Breaths/min)TimeO3RA11.522.533.50-5min 5-10min 10-15min 15-20min 20-25min 25-30minTidal Volume (L/breath)TimeO3RA 35 3.8 Pulmonary Function Response to Exercise Repeated-measures ANOVAs were utilized to analyze changes in FVC, FEV1, FEV1/FVC, PEFR, and FEF25-75 between exposure conditions (O3, Room Air) across three time points (baseline, post-CWR, post-TTE). Due to technical issues with the spirometer, baseline data for 3 participants was unusable on at least one visit. In order to include these participants in analysis, their baseline measurements from other unaffected visits were averaged. A condition-by-time interaction was reported for FVC (F(2,36) = 5.607, ηp2 = 0.238, p=0.008). Paired samples T-tests indicated that post-CWR FVC (5.54 ± 1.11L) was decreased from baseline (5.68 ± 1.09L) in the O3 condition (p=0.027), whereas no significant changes were observed between any time Table 3.2. Changes in metabolic variables during a 30 min constant work-rate cycling bout at 90% of second lactate threshold. Ozone Room Air Condition x Time Condition 170 ppb <10 ppb Interaction Main Effect Relative ?̇?O2 (ml/kg/min) 42.38 ± 6.06 42.85 ± 5.67 0.269 0.123 VCO2 (ml/min) 2672.06 ± 559.60 2670.31± 526.34 0.494 0.649 RER 0.884 ± 0.027 0.874 ± 0.030 0.848 0.03 FB (Breaths/min) 32.48 ± 7.00 31.45 ± 5.69 0.155 0.239 VT (L/breath) 2.30 ± 0.51 2.36 ± 0.46 0.147 0.279 ?̇?E (L/min) 73.24 ± 16.81 72.92 ± 14.25 0.632 0.684 ?̇?E/?̇?O2 24.29 ± 2.33 24.00 ± 2.11 0.527 0.305 ?̇?E/VCO2 27.43 ± 2.11 27.41 ± 1.78 0.392 0.978 HR (BPM) 154.6 ± 14.0 154.6 ± 14.7 0.291 0.949 36 points were observed in the room air condition (p>0.222). FVC did not differ between conditions at baseline (p=0.868) or post-TTE (p=0.188), but was lower in O3 than room air post-CWR (5.70 ± 1.11L, p=0.005). A main effect of condition was also observed for FVC (F(1,36) = 6.014, ηp2 = 0.250, p=0.025), with O3 lower than RA. No main effect of time was observed (F(2,36) = 0.306, ηp2 = 0.017, p=0.748). FEV1 also demonstrated a significant condition-by-time interaction (F(2,36) = 7.439, ηp2 = 0.292, p=0.002). Post hoc tests showed lower FEV1 at both post-CWR (4.34 ± 0.72L, 4.55 ± 0.70L, p=0.002) and post-TTE measurements (4.51 ± 0.78L, 4.61 ± 0.73L, p=0.026) in O3 compared to room air, while baseline measurement did not differ (4.48 ± 0.70, 4.50 ± 0.68, p=0.512). In the O3 condition, post-CWR FEV1 was lower than both baseline (p=0.016) and post-TTE (<0.001), with no differences between baseline and post-TTE (p=0.244). In the room air condition, FEV1 increased significantly between baseline and post-TTE (p=0.002), and between post-CWR and post-TTE (p=0.004), but not between baseline and post-CWR (p=0.164). Both a condition (F(1,36) = 9.627, ηp2 = 0.348, p=0.006) and time (F(2,36) = 8.927, ηp2 = 0.332, p<0.001) main effect were observed for FEV1, with O3 lower than RA condition, and post-TTE FEV1 was greater than post-CWR (p<0.001). No significant condition-by-time interaction (F(2,36) = 0.850, ηp2 = 0.045, p=0.436) or main effect of condition (F(1,36) = 0.909, ηp2 = 0.048, p=0.353) was observed for FEV1/FVC, although a main effect of time (F(2,36) = 7.907, ηp2 = 0.305, p=0.001) was found. FEV1/FVC was greater post-TTE (82.7 ± 6.5%) than baseline (80.8 ± 6.5%, p=0.019) and post-CWR (80.9 ± 7.3%, p=0.018), but was not different between baseline and post-CWR (p=1.000). PEFR showed a significant condition-by-time interaction (F(2,36) = 12.188, ηp2 = 0.404, p<0.001). Post hoc tests showed lower PEFR in O3 (9.11 ± 1.57L/s) compared to room air (9.86 37 ± 1.85L/s) post-CWR (p<0.001) and post-TTE (9.68 ± 1.79L/s, 10.09 ± 2.14L/s, p=0.041), while baseline was not different (9.99 ± 1.83L/s, 9.93 ± 1.84L/s, p=0.418). In the O3 condition, PEFR was lower post-CWR than both baseline (p<0.001) and post-TTE (p<0.001), but not different between baseline and post-TTE (p=0.076). In the room air condition, PEFR was greater post-TTE than post-CWR (p=0.033), but not different between baseline and post-CWR (p=0.432) or baseline and post-TTE (p=0.065). Both a condition (F(1,36) = 8.382, ηp2 = 0.318, p=0.010) and time (F(2,36) = 10.297, ηp2 = 0.364, p<0.001) main effect were observed, with PEFR in the O3 condition lower than RA, and post-CWR PEFR lower than both post-TTE (p=0.008) and baseline (p=0.007), with no difference between baseline and post-TTE (p=1.000). A significant condition-by-time interaction was observed for FEF25-75 (F(2,36) = 3.567, ηp2 = 0.165, p=0.039). FEF25-75 was lower post-CWR in the O3 condition (4.24 ± 1.01L) than room air (4.49 ± 1.02L, p=0.007), but not between baseline (p=0.677) or post-TTE (p=0.260). In the O3 condition, FEF25-75 was greatest post-TTE (4.70 ± 1.04L) compared to both baseline (4.36 ± 0.93, p=0.008) and post-CWR (p<0.001), with no differences between baseline and post-CWR (p=0.181). In the room air condition, FEF25-75 was also greatest post-TTE (4.80 ± 1.11L) compared to both baseline (4.39 ± 0.95L, p=0.002) and post-CWR (p=0.006), with no difference between baseline and post-CWR (p=0.115). A main effect of time (F(2,36) = 12.283, ηp2 = 0.406, p<0.001) but not condition (F(1,36) = 4.204, ηp2 = 0.189, p=0.055) was also observed for FEF25-75. Post-TTE FEF25-75 was greater than both baseline (p=0.008) and post-CWR (p=0.002). 38 Figure 3.6. Change in FVC (L), FEV1 (L), FEV1/FVC (%) and PEFR (L/s) with O3 exposure and exercise. * represents significantly different between conditions at a given time point (p<0.05), and ** represents a main effect of condition (p<0.05). 3456789Baseline Post-CWR Post-TTEFVC (L)Time* ** 33.544.555.566.5Baseline Post-CWR Post-TTEFEV1 (L)TimeO3RA* * ** 6065707580859095100Baseline Post-CWR Post-TTEFEV1/FVC (%)Time678910111213141516Baseline Post-CWR Post-TTEPEFR (L/s)TimeO3RA* ** * 39 Figure 3.7. Change in FEF25-75 (L/s) with O3 exposure and exercise. * represents significantly different between conditions at a given time point (p<0.05), and ** represents a main effect of condition (p<0.05). 3.9 Symptom Development with Exercise Development of symptoms was also analyzed using repeated-measures ANOVAs across baseline, post-CWR, and post-TTE. No condition-by-time interaction (F(2,38) = 2.509, ηp2 = 0.117, p=0.095), or main effect of condition (F(1,19) = 2.923, ηp2 = 0.133, p=0.104) or time (F(1.458,27.694) = 1.000, ηp2 = 0.050, p=0.357) was observed for “itchy eyes.” Similarly, no condition-by-time interaction (F(1.213,23.053) = 0.416, ηp2 = 0.021, p=0.564), or main effect of condition (F(1,19) = 0.000, ηp2 = 0.000, p=1.000) or time (F(2,38) = 2.509, ηp2 = 0.117, p=0.095) was observed for “watering eyes.” No condition-by-time interaction (F(2,38) = 1.541, ηp2 = 0.075, p=0.227), main effect of condition (F(1,19) = 0.000, ηp2 = 0.000, p=1.000) or main effect of time (F(1.253,23.811) = 1.177, ηp2 = 0.058, p=0.303) was observed for “painful or stinging sensation in eyes.” 22.533.544.555.566.57Baseline Post-CWR Post-TTEFEF 25-75 (L/s)TimeO3RA* 40 No condition-by-time interaction (F(2,38) = 3.093, ηp2 = 0.140, p=0.057), or main effect of condition (F(1,19) = 0.073, ηp2 = 0.004, p=0.789) or time (F(2,38) = 2.111, ηp2 = 0.100, p=0.135) was observed for “itchy nose.” “Blocked nose” also demonstrated no significant condition-by-time interaction (F(2,38) = 1.516, ηp2 = 0.074, p=0.233), or main effect of condition (F(1,19) = 0.101, ηp2 = 0.005, p=0.754) or time (F(2,38) = 0.463, ηp2 = 0.024, p=0.548). Similarly, no condition-by-time interaction (F(1.444,27.433) = 2.611, ηp2 = 0.121, p=0.106) or main effect of time (F(2,38) = 3.197, ηp2 = 0.144, p=0.052) was observed for “runny nose,” while a main effect of condition was present (F(1,19) = 7.795, ηp2 = 0.291, p=0.012). “Runny nose” was greater in O3 (0.7 ± 0.8) than room air (0.5 ± 0.6). No condition-by-time interaction (F(2,38) = 0.000, ηp2 = 0.000, p=1.000), or main effect of condition (F(1,19) = 0.000, ηp2 = 0.000, p=1.000) or time (F(2,38) = 0.000, ηp2 = 0.000, p=1.000) was observed for “painful or stinging sensation in nose.” “Sneezing” also demonstrated no condition-by-time interaction (F(1.430,27.179) = 2.111, ηp2 = 0.100, p=0.151), or main effect of condition (F(1,19) = 0.597, ηp2 = 0.030, p=0.449) or time (F(1.430,27.179) = 2.111, ηp2 = 0.100, p=0.151). “Shortness of breath” demonstrated a significant condition-by-time interaction (F(2,38) = 5.819, ηp2 = 0.234, p=0.006). Shortness of breath was not different at baseline between conditions (p=1.000), but was greater in O3 at both post-CWR (p=0.003) and post-TTE (p=0.017) than room air. A main effect of both condition (F(1,19) = 14.171, ηp2 = 0.427, p=0.001) and time (F(2,38) = 8.265, ηp2 = 0.513, p<0.001) were also demonstrated, with O3 (0.8 ± 0.6) greater than room air (0.4 ± 0.5), and baseline (0.1 ± 0.2) lower than both post-CWR (0.9 ± 0.8, p<0.001) and post-TTE (0.8 ± 0.7, p<0.001). “Chest tightness” also demonstrated a significant condition-by-time interaction (F(2,38) = 9.479, ηp2 = 0.333, p<0.001). Chest tightness was not different at baseline between conditions 41 (p=0.789), but was greater in O3 at both post-CWR (p=0.006) and post-TTE (p=0.005) than room air. A main effect of both condition (F(1,19) = 8.822, ηp2 = 0.317, p=0.008) and time (F(2,38) = 16.227, ηp2 = 0.461, p<0.001) were also demonstrated, with O3 (0.7 ± 0.7) greater than room air (0.2 ± 0.3), and baseline (0.1 ± 0.2) lower than both post-CWR (0.9 ± 0.5, p<0.001) and post-TTE (0.8 ± 0.6, p<0.001). No condition-by-time interaction (F(2,38) = 2.143, ηp2 = 0.101, p=0.131), or main effect of condition (F(1,19) =2.493, ηp2 = 0.116, p=0.131) or time (F(1.451,27.563) = 3.429, ηp2 = 0.153, p=0.060) was observed for “chest pain.” “Wheezing or whistling sounds in the chest” also demonstrated to significant condition-by-time interaction (F(2,38) = 2.147, ηp2 = 0.102, p=0.131) or main effect of condition (F(1,19) = 2.951, ηp2 = 0.134, p=0.102). A main effect of time was observed (F(2,38) = 3.482, ηp2 = 0.155, p=0.041), although post-hoc tests revealed no significant differences between any time points (p>0.058). “Dry, scratchy, or sore throat” also demonstrated no significant condition-by-time interaction (F(2,38) = 1.930, ηp2 = 0.092, p=0.159) or main effect of condition (F(1,19) = 0.000, ηp2 = 0.000, p=1.000). A main effect of time was observed (F(2,38) = 10.679, ηp2 = 0.360, p<0.001), wherein baseline (0.2 ± 0.4) was lower than both post-CWR (0.6 ± 0.7, p=0.001) and post-TTE (0.6 ± 0.6, p=0.002). No condition-by-time interaction (F(2,38) = 2.304, ηp2 = 0.108, p=0.114), or main effect of condition (F(1,19) =0.000, ηp2 = 0.000, p=1.000) was observed for “headache,” while a main effect of time (F(2,38) = 3.827, ηp2 = 0.168, p=0.031) was significant. Post-hoc tests revealed no significant differences between any time points (p>0.073). “Fatigue” demonstrated a significant condition-by-time interaction (F(2,38) = 3.924, ηp2 = 0.171, p=0.028). Fatigue was not different at baseline between conditions (p=0.309), but was 42 greater in O3 at both post-CWR (p=0.038) and post-TTE (p=0.002) than room air. A main effect of both condition (F(1,19) = 8.635, ηp2 = 0.312, p=0.008) and time (F(2,38) = 5.945, ηp2 = 0.238, p=0.006) were also demonstrated, with O3 (1.1 ± 0.9) greater than room air (0.6 ± 0.6), and baseline (0.6 ± 0.6) lower than both post-CWR (1.0 ± 0.9, p=0.037) and post-TTE (1.0 ± 0.8, p=0.026). No condition-by-time interaction (F(1.447,27.491) = 0.222, ηp2 = 0.012, p=0.730), or main effect of condition (F(1,19) =0.681, ηp2 = 0.035, p=0.419) or time (F(1.432,27.207) = 1.498, ηp2 = 0.073, p=0.239) was observed for “nausea.” “Dyspnea” demonstrated a significant condition-by-time interaction (F(2.839,51.107) = 6.802, ηp2 = 0.274, p=0<0.001). Dyspnea did not differ at baseline (p=0.716), 10 mins into CWR (p=0.397), 20 mins into CWR (p=1.000), or 30 mins into CWR (p=0.087), while dyspnea was higher in O3 at both post-CWR (p=0.004) and post-TTE (p=0.002). Main effects of both condition (F(1,18) = 4.787, ηp2 = 0.210, p=0.042) and time F(2.069,37.235) = 16.201, ηp2 = 0.474, p<0.001) were also present, with O3 (1.4 ± 1.0) greater than room air (1.1 ± 0.8), and dyspnea increasing between all adjacent time points between baseline and 30 mins into CWR (p<0.024), before decreasing at post-CWR and post-TTE. 43 Figure 3.8. Change in symptom development with O3 exposure and exercise. Results are presented as mean ± standard deviation. * represents significantly different between conditions at a given time point (p<0.05), and ** represents a main effect of condition (p<0.05). -0.500.511.522.5Baseline Post-CWR Post-TTEChest Tightness (0-5)Time** * * -0.500.511.522.53Baseline Post-CWR Post-TTEShortness of Breath (0-5)Time* * ** -0.500.511.522.533.5Baseline Post-CWR Post-TTEFatigue (0-5)TimeO3Room Air** * * 44 Figure 3.9. Change in dyspnea development with O3 exposure and exercise. Results are presented as mean ± standard deviation. * represents significantly different between conditions at a given time point (p<0.05), and ** represents a main effect of condition (p<0.05). 3.10 Time-to-Exhaustion Test Exercise Response Two-tailed, paired-samples T-tests were utilized to compare metabolic data between O3 and room air conditions for the time-to-exhaustion test, with data presented as an average for both the last minute, and last shared minute ± standard deviation. The last minute refers to the average of the last 60 seconds for the TTE test for each condition, whereas the last shared minute refers to the last 60 seconds shared between TTE tests between conditions. As maximal exercise values are heavily reliant on each participant delivering their highest effort, changes in effort between trials have the potential to confound results. A Grubbs test for statistical outliers for change in TTE in seconds between the O3 and room air conditions identified a single outlier (z-score: 2.69, p<0.05), as he exercised for 142 seconds longer in O3 than room air. As such, results for the last minute of the TTE are presented as both including and excluding this participant -0.500.511.522.533.544.5Baseline 10min CWR 20min CWR 30min CWR Post-CWR Post-TTEDyspnea (0-10)TimeO3RA* ** * 45 (participant 4). A further participant (participant 8) encountered musculoskeletal pain in the knee upon starting the TTE on his 3rd experimental visit, so did not complete either TTE test and is therefore excluded from the analysis. When including all participants, during the last minute of the TTE, relative ?̇?O2 was lower (-2.01 ± 2.59 ml/kg/min, CI: [-3.26, -0.76]) in O3 (59.01 ± 7.13 ml/kg/min) than in the room air (61.02 ± 6.94 ml/kg/min) condition (t = -3.83, ηp2 = 0.449, p=0.003). VCO2 (t = -1.268, ηp2 = 0.082, p=0.221), RER (t = 2.031, ηp2 = 0.186, p=0.057), and breathing frequency (t = 0.224, ηp2 = 0.003, p=0.825) were not different between conditions. Tidal volume was significantly lower (-0.10 ± 0.15 L/breath, CI: [-0.17, -0.03) in the O3 condition (2.73 ± 0.58 L/breath) than in room air (2.83 ± 0.58 L/breath, t = -3.014, ηp2 = 0.335, p=0.007). Minute ventilation was also significantly lower (-4.72 ± 9.63 L/min, CI: [-9.36, -0.07) in the O3 condition (144.36 ± 31.21 L/min) than in room air (149.08 ± 30.39 L/min, t = -2.134, ηp2 = 0.202, p=0.047). Both ?̇?E/?̇?O2 (t = 0.079, ηp2 = 0.000, p=0.938) and ?̇?E/VCO2 (t = -1.816, ηp2 = 0.155, p=0.086) did not differ between conditions, whereas heart rate was significantly lower (-4 ± 7 bpm, CI: [-7, 0) in the O3 condition (176 ± 12 bpm) than in room air (179 ± 13, t = -2.322, ηp2 = 0.252, p=0.034). Time-to-exhaustion in seconds was not different between conditions (t = -1.779, ηp2 = 0.150, p=0.092). Results remained the same regardless of whether participant 4 was removed from subsequent analysis during the last minute of the TTE for relative ?̇?O2, VCO2, tidal volume, breathing frequency, ?̇?E/?̇?O2, ?̇?E/VCO2, and heart rate. When participant 4 was removed, RER became greater (0.02 ± 0.04, CI: [0.00, 0.04]) in O3 (1.11 ± 0.05) than in the room air (1.09 ± 0.05) condition (t = 2.342, ηp2 = 0.244, p=0.032). Minute ventilation was no longer significantly lower in O3 than room air when participant 4 was excluded from analysis (t = -2.026, ηp2 = 46 0.194, p=0.059). With removal of participant 4, TTE in seconds was lower (-35 ± 49 seconds, CI: [-59, -11]) in O3 (204 ± 55 seconds) than in the room air (238 ± 82 seconds) condition (t = -3.032, ηp2 = 0.351, p=0.008). Figure 3.10. Change in relative ?̇?O2 and VCO2 in the last minute of the TTE. * represents significantly different between conditions with all participants and removal of participant 4 (p<0.05), ** represents a significant difference only when participant 4 was removed (p<0.05), and *** represents a significant difference only when all participants were included (p<0.05). 4045505560657075Relative 𝑉̇O2 (ml/kg/min)O3 Room Air* 30003500400045005000550060006500VCO2O3 Room Air 47 Figure 3.11. Change in RER, ?̇?E, FB, and VT in the last minute of the TTE. * represents significantly different between conditions with all participants and removal of participant 4 (p<0.05), ** represents a significant difference only when participant 4 was removed (p<0.05), and *** represents a significant difference only when all participants were included (p<0.05). 0.90.9511.051.11.151.2Respiratory Exchange RatioO3 Room Air** 80100120140160180200220Minute Ventilation (L/min)O3 Room Air*** 35404550556065707580Breathing Frequency (breaths/min)O3 Room Air** 1.522.533.544.5Tidal Volume (L/breath)O3 Room Air* 48 Figure 3.12. Change in ?̇?E/?̇?O2, ?̇?E/VCO2, HR, and TTE in the last minute of the TTE. * represents significantly different between conditions with all participants and removal of participant 4 (p<0.05), ** represents a significant difference only when participant 4 was removed (p<0.05), and *** represents a significant difference only when all participants were included (p<0.05). 283032343638404244𝑉̇E/𝑉̇O2O3 Room Air2527293133353739𝑉̇E/VCO2O3 Room Air150160170180190200210HR (bpm)O3 Room Air*75125175225275325375425475TTE (seconds)O3 Room Air** 49 Table 3.3. Changes in metabolic variables during the last minute of a time-to-exhaustion cycling bout at 105% of peak power. 170ppb O3 Room Air P-Value Relative V̇O2 (ml/kg/min) 59.01 ± 7.13 61.02 ± 6.94 0.003 VCO2 (ml/min) 4648.28 ± 882.03 4722.64 ± 879.30 0.221 RER 1.110 ± 0.053 1.089 ± 0.045 0.057 FB (Breaths/min) 53.56 ± 9.14 53.33 ± 8.80 0.825 VT (L/breath) 2.73 ± 0.58 2.83 ± 0.58 0.007 V̇E (L/min) 144.36 ± 31.21 149.08 ± 30.39 0.047 V̇E/V̇O2 34.39 ± 3.37 34.35 ± 2.66 0.938 V̇E/VCO2 31.01 ± 2.90 31.57 ± 2.68 0.086 HR (BPM) 175.6 ± 12.0 179.4 ± 12.9 0.034 During the last shared minute of the TTE between conditions, relative ?̇?O2 was lower (-2.04 ± 1.66 ml/kg/min, CI: [-2.84, -1.24]) in O3 (58.45 ± 7.28 ml/kg/min) than in the room air (60.49 ± 6.96 ml/kg/min) condition (t = -5.370, ηp2 = 0.616, p<0.001). VCO2 (t = -2.027, ηp2 = 0.186, p=0.058), RER (t = 1.556, ηp2 = 0.119, p=0.137), breathing frequency (t = 2.019, ηp2 = 0.185, p=0.059), and minute ventilation (t = -0.427, ηp2 = 0.010, p=0.674) were not different between conditions. Tidal volume was significantly lower (-0.22 ± 0.25 L/breath, CI: [-0.34, -0.10) in the O3 condition (2.77 ± 0.58 L/breath) than in room air (2.99 ± 0.59 L/breath, t = -3.793, ηp2 = 0.444, p=0.001). ?̇?E/?̇?O2 (t = 1.295, ηp2 = 0.085, p=0.212), ?̇?E/VCO2 (t = 0.726, ηp2 = 0.028, p=0.477), and heart rate (t = -2.116, ηp2 = 0.230, p=0.052) did not differ between conditions. 50 Figure 3.13. Change in relative ?̇?O2, VCO2, RER, and ?̇?E in the last shared minute of the TTE. * represents significantly different between conditions (p<0.05). 4045505560657075Relative 𝑉̇O2 (ml/kg/min)O3 Room Air*3000350040004500500055006000VCO2O3 Room Air0.930.981.031.081.131.181.23Respiratory Exchange RatioO3 Room Air80100120140160180200220Minute Ventilation (L/min)O3 Room Air 51 Figure 3.14. Change in FB, VT, ?̇?E/?̇?O2, and ?̇?E/VCO2 in the last shared minute of the TTE. * represents significantly different between conditions (p<0.05). 354045505560657075Breathing Frequency (breaths/min)O3 Room Air1.522.533.544.5Tidal Volume (L/breath)O3 Room Air*283032343638404244𝑉̇E/𝑉̇O2O3 Room Air242628303234363840𝑉̇E/𝑉̇CO2O3 Room Air 52 Figure 3.15. Change in HR in the last shared minute of the TTE. * represents significantly different between conditions (p<0.05). Table 3.4. Changes in metabolic variables during the last shared minute of a time-to-exhaustion cycling bout at 105% of peak power. 170ppb O3 Room Air P-Value Relative V̇O2 (ml/kg/min) 58.45 ± 7.28 60.49 ± 6.96 <0.001 VCO2 (ml/min) 4614.26 ± 922.04 4720.90 ± 918.05 0.058 RER 1.109 ± 0.051 1.095 ± 0.044 0.137 FB (Breaths/min) 52.04 ± 10.47 48.47 ± 7.87 0.059 VT (L/breath) 2.77 ± 0.58 2.99 ± 0.59 0.001 V̇E (L/min) 141.97 ± 33.53 143.21 ± 30.59 0.674 V̇E/V̇O2 33.99 ± 3.72 33.21 ± 2.91 0.212 V̇E/VCO2 30.66 ± 3.18 30.32 ± 2.45 0.477 HR (BPM) 176.2 ± 12.7 178.3 ± 11.1 0.052 150160170180190200HR (bpm)O3 Room Air 53 Chapter 4: Discussion 4.1 Summary This thesis study aimed to investigate the effects of real-world levels of O3 exposure on pulmonary function, metabolic variables, symptom development, and exercise capability in highly-trained endurance athletes. Twenty participants completed a double-blind, randomized crossover design wherein they performed submaximal and maximal exercise during room air (<10 ppb O3) and O3 (170 ppb) conditions. We demonstrated no changes between conditions during extended exercise in the moderate and heavy intensity domains, while metabolic variables and breathing pattern during maximal exercise in the severe intensity domain were significantly altered during O3 exposure, with trends towards lower time to exhaustion. We also observed significantly reduced pulmonary function and increased development of respiratory symptoms following O3 exposure and exercise, and following resting exposure to 750 ppb O3. These findings indicate a strong likelihood of an O3-induced diminished oxygen transport due to changes in ventilatory pattern, and decreased motivation to perform maximal exercise due to strong respiratory symptoms. Understanding the mechanisms of altered physiological responses to exercise during O3 exposure is essential for mitigating performance changes in athletes during high-pollution athletic scenarios. 4.2 Response during Submaximal Exercise As expected, no changes in any variables were observed between conditions for the 25-minute warmup exercise bout at 90% of 1st lactate threshold. This is likely due to the lower exercise intensity, as well as the time-course required for negative effects of O3 exposure to develop. Previous studies have not demonstrated a change in anaerobic threshold under O3 concentrations of up to 300 ppb, indicating that O3 may only provide a ventilatory limitation, as 54 opposed to other indirect effects on oxygen transport (Savin & Adams, 1979). As the exercise intensity for the warmup bout was designed to be slightly below 1st lactate threshold (aerobic threshold), and in the moderate intensity domain, where ventilation does not limit exercise capability, these results indicate further support for an effect of intensity. Additionally, although the time course required for negative effects of O3 to occur is not fully understood, previous research points to an added effect when effective dose is increased by extending the time of exposure. When pulmonary function was measured halfway through a 2-hour intermittent exercise protocol with O3 exposure, changes were approximately half of those observed upon completion of the protocol (Silverman et al., 1976). As participants in this thesis study began exposure to the experimental condition at the beginning of the warmup exercise bout, it was unlikely that any notable changes would occur within the 25-minutes. However, ensuring that intensity (i.e., ?̇?O2, RER, HR, blood lactate), as well as effective dose of pollution (based on ?̇?E) were matched between exposure conditions assists in avoiding confounding results of subsequent higher intensity bouts of exercise. Analysis of the 30-minute exercise bout at 90% of 2nd lactate threshold showed no significant differences between conditions, apart from RER. A condition main effect indicated that RER was greater in the O3 condition (0.88 ± 0.06) than in room air (0.87 ± 0.07). If O3 were to influence substrate utilization by limiting O2 transport, significant ramifications may be had for athletic performance, particularly in longer duration activity. A previous study demonstrated greater RER during the last 30-minutes of a 2-hour exposure to O3 than in a filtered air condition, although did not attempt to explain this finding (Horvath et al., 1979). Additionally, these changes were in the magnitude of 0.05 (250 ppb) to 0.08 (750 ppb) (Horvath et al., 1979). While the ParvoMedics TrueOne 2400 metabolic cart system has demonstrated low technological error 55 and excellent inter-unit repeatability, absolute percent error for ?̇?O2 and VCO2 were calculated at 2.1% (MacFarlane & Wu, 2013). Therefore, in the absence of other supporting evidence (i.e., differences in ?̇?O2 or VCO2), the small (0.01) change in RER observed between O3 and room air conditions was not considered material. While it was expected that ?̇?O2 would be matched between conditions given an identical external workload, the researchers expected changes in ventilatory pattern as a result of O3 exposure, particularly towards the end of the exercise bout. Previous studies examining continuous exercise have demonstrated dose-dependent changes in ventilatory pattern, namely, an increase in breathing frequency and decrease in tidal volume (Lauritzen & Adams, 1985). These changes were only significant after 60 minutes of exercise at 300 ppb and 400 ppb, with no changes at 200 ppb (Lauritzen & Adams, 1985). Other studies involving intermittent exercise mirrored these findings of changes in frequency and depth of breathing, citing O3 stimulation of irritant receptors in the lungs (McDonnell et al., 1983). However, this result was again found in a longer protocol (2 hours) with higher O3 concentrations (240 ppb and greater), with no changes at 180 ppb or lower (McDonnell et al., 1983). While the high fitness level of the participants in this thesis study increased effective pollution dose due to increased sustainable minute ventilation, it was clearly below the threshold for changes in metabolic variables. These previous studies indicate that there is a strong dose-response relationship in ventilatory pattern changes with O3 exposure. Although we did not demonstrate a change in ventilatory pattern, visual inspection of the data (Figure 3.4 & 3.5) suggests that differences between conditions became greater towards the end of the 30-minute exercise bout, even if they were not statistically significant. If participants were asked to maintain the workload for longer than 30 minutes, it is possible that significant 56 changes would be observed due to the observed time course for alterations in breathing pattern (Schelegle et al., 2007). However, it is unlikely that anaerobic threshold itself would be shifted, as even when ?̇?O2max was limited during O3 exposure, no changes to anaerobic threshold were observed (Savin & Adams, 1979). As O3 exposure is thought to only impose a ventilatory limitation during exercise, as opposed to negative impacts on oxygen transport in the arterial blood or at the mitochondria (Savin & Adams, 1979), it seems unlikely that changes in ability to perform exercise when ventilation is not limited (i.e., in the heavy exercise domain) would be experienced. This corresponds with the data obtained from this thesis study, wherein we showed no changes in blood lactate, rating of perceived exertion, or ?̇?E/?̇?O2 between conditions throughout the 30-minute exercise bout slightly below 2nd lactate threshold, which all would be expected to change when exercise intensity reaches the severe domain. 4.4 Response during Maximal Exercise In contrast to previous submaximal exercise bouts (25 mins at 90% of LT1, 30 mins at 90% of LT2), the additional stress of maximal exercise significantly impacted metabolic variables under O3 exposure. Analysis of the TTE test demonstrated clear differences between O3 and room air conditions, both when examining the last minute of exercise in each condition, as well as the last shared minute of exercise between conditions. End-test values (last minute) will be influenced heavily by the overall exercise duration due to both ?̇?O2 onset kinetics and development of the ?̇?O2 slow component (Jones et al., 2011). As exercise duration may be skewed not only by physiological mechanisms but also by participant effort, comparison of the same time period (last shared minute) between conditions mitigates these confounding variables and provides more clarity as to the physiological responses. 57 As previously explained, O3 exposure activates irritant receptors in the respiratory tract, causing both local and autonomic changes to limit pollution exposure (ISA for Ozone and Related Photochemical Oxidants, 2020). While these changes may be adaptive from a health perspective, they likely inhibit exercise capability. O3 exposure is unlikely to impact residual volume during exercise, but instead it likely reduces the ability to perform deep inspirations to total lung capacity, resulting in an adjusted breathing pattern (Gibbons & Adams, 1984). Although these changes were only present during maximal exercise in this study, a shift in breathing pattern towards shallower, more frequent breaths under O3 exposure during exercise is well-documented in previous literature. Lower tidal volume under O3 exposure has been observed during maximal exercise following submaximal exercise (Folinsbee et al., 1975, 1977; Gong et al., 1986), and extended submaximal exercise itself (Gibbons & Adams, 1984; McDonnell et al., 1983). However, other studies where the exposure time was reduced, such as with a single graded cycling test to exhaustion (Savin & Adams, 1979), showed only non-significant trends towards lower tidal volume. This indicates that there is a combined effect of overall “effective dose” of pollution, as well as exercise intensity on the severity of responses (Delucia et al., 1977). As the time-to-exhaustion test was performed following 55-minutes of submaximal exercise, it is challenging to elucidate whether effects were observed due to the increased exercise intensity imposing a greater stress, or that the effective dose of pollution was greater at this time. However, as only a few minutes of exercise (and therefore pollution exposure) separated the last 5 minutes of the constant work-rate bout, wherein no changes were observed in breathing pattern, and the TTE test, where significant changes in breathing pattern were observed, it seems likely that increased exercise intensity played a significant role. 58 During the last shared minute of the TTE, tidal volume was significantly lower, and breathing frequency trended non-significantly towards being higher (p=0.059) in O3, resulting in a matched minute ventilation between conditions. A similar trend was observed in the last minute of the TTE, with lower tidal volume in O3 with no change in breathing frequency, while minute ventilation was lower (-4.72 ± 9.63 L/min). Other studies have also noted lower minute ventilation while also reporting lower tidal volume under O3 exposure (Gong et al., 1986). While lower minute ventilation can obviously negatively impact exercise capability, the adopted breathing pattern is also highly inefficient, and likely had physiological consequences. Although minute ventilation is the product of the depth and number of breaths taken per minute, it is measured at the whole-body level and does not represent alveolar ventilation. As oxygen must travel through the conducting airways to reach the alveoli to undergo gas exchange, it is inevitable that some air will remain in the conducting airways (trachea, bronchi) and not reach the alveoli, which is known as anatomical deadspace (Ferluga et al., 2018). Anatomical deadspace is stature dependent, but generally equates to 150-200mL in healthy individuals, corresponding to about 30% of tidal volume at rest (McArdle et al., 2015). Further losses in gas exchange may occur when inadequate perfusion is delivered to well-ventilated alveoli, or with impairments to diffusion, shunt, or increased alveolar ventilation to cardiac output ratio (Robertson, 2015). As these factors occur in areas where gas exchange can occur (i.e., not conducting airways), this is referred to as physiological deadspace (Ferluga et al., 2018). However, clear changes in breathing pattern are observed with O3 exposure, which impacts the proportion “lost” to anatomical deadspace, and causes concomitant reductions in alveolar ventilation (Folinsbee et al., 1975). 59 Few studies have directly measured alveolar ventilation under O3 exposure. During a 60-minute submaximal cycling protocol, participants demonstrated lower alveolar ventilation, likely due to the increased breathing frequency and decreased tidal volume observed (Gibbons & Adams, 1984). A similar protocol in runners also showed changes in breathing pattern during a 60-min exercise bout exposed to O3, however, no changes in alveolar ventilation were noted (Adams & Schelegle, 1983). Studies in clinical populations have demonstrated that in patients with cystic fibrosis, increased deadspace ventilation is caused by increased respiratory frequency, and likely alters exercise capability (Thin et al., 2004). Despite mixed evidence, it is logical that even if minute ventilation is matched by increasing breathing frequency and decreasing tidal volume, then a greater percentage of each breath must be lost to deadspace and alveolar ventilation would be lower. With less oxygen available for diffusion across the alveoli, lower arterial oxygen concentrations would occur, resulting in less oxygen available for use at the working muscles. However, without direct measurement of alveolar ventilation, it is challenging to make definite conclusions. It is also possible that O3-induced pulmonary edema or inflammation can cause significant reductions in diffusion across the alveolar membrane. Due to the toxic properties of O3, significant oxidative stress, injury, and inflammation can occur in the respiratory tract (ISA for Ozone and Related Photochemical Oxidants, 2020). Pulmonary edema develops when the rate of fluid removal by the lymphatic system is unable to match the fluid movement into the lung interstitium, causing a buildup of extravascular lung fluid (Iles et al., 2009). Previous studies in animal models have demonstrated massive pulmonary edema under high concentrations of O3 exposure, often causing death (Matzen, 1957). As alveolar flooding due to increased extravascular fluid limits gas exchange by thickening the barrier across which 60 diffusion occurs (Iles et al., 2009), if this situation were to occur in humans during exercise, it is logical to assume that arterial blood would become less saturated with oxygen, reducing availability at the tissues. Other extreme environmental conditions can also impact gas exchange at the alveoli. In high altitude pulmonary edema, significantly decreased oxygen saturations measured via pulse oximetry are observed in patients compared to unaffected controls (Hultgren et al., 1996), demonstrating the ability of fluid buildup to impact diffusion. Previous studies in humans have demonstrated lower diffusion capacity under high levels of O3 exposure (600-800 ppb), as measured by the diffusing capacity for carbon monoxide (DLCO) (Young et al., 1964). The previous study also noted that due to the fairly short time course for recovery, it was likely that pulmonary edema was the primary cause, rather than an inflammatory response, and that effects were likely to be largest in the most well-ventilated sections of the lungs (Young et al., 1964). Whether real-world levels of O3 exposure can cause severe enough pulmonary edema to limit diffusion across the membrane and alter physiological deadspace is currently unknown. While the short time course for pulmonary function and symptom recovery in this thesis study supports the potential for transient pulmonary edema, neither DLCO nor bronchoalveolar lavage were performed in testing. Due to a combination of an inefficient breathing pattern and potential reduction in diffusion capacity, it seems likely that arterial oxygen content was lower in the O3 condition, reducing the amount available to the working muscles during maximal exercise. We demonstrated significantly lower ?̇?O2 in O3 compared to room air, both during the last minute of the TTE, as well as the last shared minute between each condition. If a reduction in ?̇?O2 was only observed in the last minute of exercise, it would be possible that differences were solely due to a lack of participant effort causing shorter exercise time, and was therefore influenced by ?̇?O2 61 kinetics. Previous studies that have demonstrated lower ?̇?O2 under O3 conditions typically only report peak values, increasing the potential for confounding variables to influence results (Folinsbee et al., 1977; Gong et al., 1986). However, we demonstrated nearly identical changes between the last minute (-2.01 ± 2.59 ml/kg/min) and the last shared minute (-2.04 ± 1.66 ml/kg/min) in O3 compared to room air, indicating greater likelihood of a physiological mechanism limiting oxygen consumption during maximum exercise. It remains widely accepted that in fit individuals under normobaric conditions, ?̇?O2max is limited centrally rather than peripherally, largely due to cardiac output (Wagner, 2000). Therefore, if arterial oxygen concentration were to be decreased during O3 exposure, lower O2 content would be delivered to the tissue, irrespective of changes in cardiac output. This would in turn reduce the capacity for exercise, and cause the observed significant reductions in time-to-exhaustion during the maximal test (-35 ± 49 seconds) with the removal of participant 4. Twelve of nineteen participants who completed the TTE test demonstrated lower TTE in O3 than room air. It is also possible that symptom development also contributed to lower exercise time in O3 conditions. Previous studies have documented this concern, indicating that participants were likely limited by respiratory symptoms that prevented them from providing a true maximal effort (Folinsbee et al., 1977). However, the presence of reduced ?̇?O2 achieved during the last shared minute of the TTE indicates that both physiological and psychological variables play a strong role in the ability to perform exercise during O3 exposure. 4.5 Symptom Development with Exercise Four symptoms demonstrated significant condition-by-time interactions when measured throughout experimental trials: chest tightness, shortness of breath, fatigue, and dyspnea. These symptoms showed no difference in symptom severity at baseline, but were higher in O3 than 62 room air when measured post-CWR and post-TTE. Dyspnea, which was also measured every 10-minutes throughout the CWR exercise bout in addition to baseline and post-exercise values was not different between conditions during exercise. These symptoms have been previously reported, and the timing of their onset indicates the importance of the length of pollution exposure. A previous systematic review examining O3 exposure during exercise strongly indicated the increased likelihood and severity of symptom development (Hung et al., 2021). Of a variety of symptoms, respiratory symptoms of dyspnea, cough, pain during deep inhalation, and wheezing were most observed (Hung et al., 2021). While we did not assess “cough” severity as part of our symptom questionnaire, most participants experienced coughing following exercise protocols in the O3 condition, particularly when performing spirometry. These symptoms are likely due to the irritant receptor response within the respiratory tract (ISA for Ozone and Related Photochemical Oxidants, 2020), causing participant discomfort. As the measured symptoms became more severe in the O3 condition following the constant work rate bout, and remained higher following the time-to-exhaustion test, it is likely that participant performance was affected in part by the presence of negative respiratory symptoms. Numerous previous studies have indicated symptom development as a potential contribution to reduced exercise capability under O3 exposure. While it is challenging to elucidate the role of psychological versus physiological mechanisms, studies have indicated that subjective symptoms may have affected exercise during competitive simulations (Adams & Schelegle, 1983), graded exercise tests to maximum (Folinsbee et al., 1977), and continuous moderate exercise (Gibbons & Adams, 1984). It is likely that the presence of uncomfortable respiratory symptoms experienced prior to the time-to-exhaustion test likely impacted participant 63 readiness and motivation, impacting exercise performance. However, due to the wide range of interindividual variability in the development of O3-related respiratory symptoms, it is likely that some participants were affected more than others. Although the study was double-blinded to the exposure condition, the development of adverse respiratory symptoms assisted participants in effectively determining which exposure condition they had received during the cross-over design. Only eleven of twenty participants were able to correctly identify their exposure condition order with a confidence rating greater than 2.5/5 upon completion of the study. However, these participants were generally those who experienced stronger adverse symptoms related to O3 exposure. Previous studies examining diesel exposure have showed that symptoms are better related to an individual’s perception of the exposure condition, rather than the exposure itself (Carlsten et al., 2013). Other studies have also demonstrated increased ratings of dyspnea when participants were deceived to believe that they were breathing hypoxic gas mixture, indicating the effect of expectations in feelings of breathlessness (Kipp et al., 2022). As a result, it is possible that development of strong respiratory symptoms which aided participants in determining the air condition during trials, in combination with expectancy as to the effects of O3 exposure may have influenced participant effort during testing. 4.6 Pulmonary Function Changes with Exercise Predictably, O3 exposure during exercise negatively impacted pulmonary function measures, with lower FVC, FEV1, PEFR, and FEF25-75 in O3 than room air post-CWR. FEV1 and PEFR remained lower post-TTE in O3 than room air, while no differences between conditions were observed for FEV1/FVC. As pulmonary function was generally the lowest post-CWR, and 64 tended to recover following the TTE test, there is likely an interplay between the bronchodilatory effects of maximal exercise, and the negative impacts of O3 exposure. A reduction in pulmonary function in response to O3 exposure during exercise is well documented in previous literature. A systematic review of randomized control trials indicated strong evidence for O3 exposure to negatively impact spirometry following moderate-to-vigorous exercise (Hung et al., 2021). However, the magnitude of responses can vary greatly depending on the effective dose of pollution delivered during the study, which is impacted by not only the O3 concentration, but exposure length, intensity, and population group, amongst others. When elite cyclists were exposed to O3 at 120 ppb and 200 ppb during a 60-minute submaximal exercise bout followed by an incremental test to exhaustion, decreases in FEV1 compared to filtered air were 5.6% and 21.6%, respectively, while decreases in FVC were 7.6% and 19.1% (Gong et al., 1986). The largest change in FEV1 under 170 ppb O3 observed in this thesis study was 0.21L (4.6%), which is like that of 120 ppb exposure in Gong et al., but much smaller than observed at 200 ppb, whereas the largest change in FVC was 2.8%. These differences may be driven by slightly higher minute ventilations maintained (89 L/min in Gong et al.), as the exposure duration was similar. Other studies examining pulmonary function responses to O3 exposure have noted similar results, with a 5.8% decrease in FEV1 and 7.8% decrease in FVC following a competitive cycling simulation at 180 ppb, with a much larger decrease (FEV1: 10.5%, FVC: 9.9%) when O3 concentration was increased to 240 ppb (Schelegle & Adams, 1986). These results support a previously proposed sigmoidal shape of pulmonary function responses to O3, if the changes in effective dose occur on the steep portion of the curve (McDonnell et al., 1983).While nearly all studies describe changes in FVC and FEV1, few studies have reported peak expiratory flows or mid-expiratory flows. Sixty minutes of 65 submaximal cycling in 150 ppb and 300 ppb O3 created changes of 0.09 L and 0.67 L in FEF25-75, respectively (Gibbons & Adams, 1984), while we observed a difference of 0.25 L post-CWR between O3 and room air. Decreases in FVC and FEV1 are largely related to the ability to maximally inflate the lungs, and function of the large airways upon forceful expiration (Kim et al., 2021). Due to activation of irritant receptors within the respiratory tract under O3 exposure, decreased total lung capacity is commonly observed without changes in residual volume, indicating a decrease in inspiratory capacity (Gibbons & Adams, 1984; Messineo & Adams, 1990). Incomplete filling of the lungs also reduces the static recoil pressure (Mead et al., 1966), which likely contributed to the lower FEV1 and PEFR observed in this study. Further inflammation and bronchoconstriction due to O3 exposure would increase flow resistance (ISA for Ozone and Related Photochemical Oxidants, 2020), and cause the observed changes in FEF25-75, which is less likely to be impacted by maximum flows and represents the medium to small airways (Kim et al., 2021). Despite the increased effective dose of pollution following all exercise tests, pulmonary function tended to recover slightly following the TTE test. Only FEV1 and PEFR remained lower in O3 than in room air in the post-TTE measurement. While healthy individuals tend to be well bronchodilated even at rest, exercise can provide further bronchodilatation of the airways, with higher ventilations a key contributor to changes in the airways (Gotshall, 2006). It is likely that increased bronchoconstriction by O3 exposure was partially mitigated by the bronchodilatory effects of maximal exercise in healthy individuals. In the room air condition of this study, FEV1 did not change between baseline and post-CWR (55 mins of submaximal exercise), but did increase post-TTE. Despite the protective effects of exercise-induced bronchodilation on O3 exposure, clear changes in ventilatory pattern were observed throughout the time-to-exhaustion 66 test. It is possible that including a higher-intensity exercise bout as part of warm-up protocols might stimulate increased bronchodilation and mitigate negative impacts of O3 exposure. Resting O3 Pulmonary Function Response Predictably, a 2-hour resting exposure to 750 ppb O3 resulted in large decreases in pulmonary function and increases in participant discomfort, both through increases in negative symptoms and development of dyspnea. Although the O3 concentration is greater than that seen in the ambient environment, previous laboratory studies examining O3 exposure and exercise have also used this concentration. Previous similar exposures examples at 750 ppb include 2-hours of rest (Horvath et al., 1979), and 2-hours of intermittent periods of rest and exercise (Folinsbee et al., 1977; Silverman et al., 1976). Average decreases in pulmonary function are comparable between these studies and the results of this thesis. Utilizing only resting O3 exposure at 250 ppb, 500 ppb, and 750 ppb, Horvath et al. showed decreased FVC, FEV1, FEV2, and FEV3 in both the 500 ppb and 750 ppb conditions compared to filtered air. These differences in pulmonary function were approximately 5% at 500 ppb, and 9-10% at 750 ppb (Horvath et al., 1979). Folinsbee et al. demonstrated that after 2-hours of alternating 15-minutes of rest with 15-minutes of light cycling exercise (~50 watts), FVC and FEV1 were 81.8% and 77.2% of the values observed in filtered air. Symptoms of exposure included irritation of the throat, discomfort upon deep inspiration, coughing, and occasionally nausea (Folinsbee et al., 1977). Similarly, Silverman et al. exposed participants to a range of O3 concentrations, including 750 ppb, during both rest and intermittent exercise. FVC was 0.24 L lower in 0.75ppm O3 compared to filtered air during rest, and 0.53 lower in O3 under intermittent exercise, although neither result reached statistical significance. However, FEV1 was significantly lower in both rest (0.32 L) and intermittent exercise (0.84 L) when compared to 67 identical trials in filtered air (Silverman et al., 1976). The results of these studies clearly show significant reductions in pulmonary function as a result of high effective doses of O3, with greater decreases when participants are exposed to higher concentrations or minute ventilation is increased due to an exercise stimulus. Although ventilation was not measured during resting O3 exposure in our study, it can be estimated based on typical resting ventilation values, O3 concentration, and duration of exposure. Based on an estimated resting ventilation of 8L/min (Horvath et al., 1979), 750 ppb O3 concentration, and 120 minutes of exposure, effective dose can be estimated at 720 AU. A decrease in FVC of 0.52L immediately post-exposure, and 0.33L 30 mins post exposure compared to baseline measurement in this study may be due to an increased residual volume, decreased total lung capacity, or a combination of both factors (Silverman et al., 1976). Although absolute lung volumes were not measured directly, previous studies have demonstrated potential contributions of both, with greater support for decreased total lung capacity. A previous study examining pulmonary function following exercise in 300 ppb O3 did not demonstrate a change in residual volume, but significant decreases in FVC and total lung capacity (Gibbons & Adams, 1984). Other studies with similar protocols also showed no change in residual volume, and indicated that decreased FVC was attributed to decreased maximum inspiratory volume (Messineo & Adams, 1990). These effects are likely due to the stimulation of irritant receptors within the respiratory tract, causing a limitation of full inspiration (Gibbons & Adams, 1984). Activation of certain sensory nerves, such as C-fibers, can cause local reflex responses to limit maximal inspiration to protect the ventilatory system from further exposure to the pollutant (ISA for Ozone and Related Photochemical Oxidants, 2020). Previous studies have indicated that changes in vital capacity are likely unrelated to decreased lung compliance, which would reduce 68 total lung capacity, but rather an inhibition of maximal inspiration due to strong discomfort. Bates et al. demonstrated that a decreased maximum static elastic recoil pressure of the lung following O3 exposure occurred without a decrease in static lung compliance (Bates et al., 1972). This indicates that lung compliance is unaffected by O3 exposure, and that decreases in FVC are likely caused by a reduction in inspiratory effort due to discomfort (Bates et al., 1972). As the maximum expiratory flow rate is determined by the static recoil pressure and the flow-resistance of upstream airways, a reduction in static recoil pressure due to incomplete filling of the lungs will also contribute to the reduction in flow rates (Mead et al., 1966). The activation of irritant receptors within the lungs can be further supported by the development of symptoms accompanying reduced pulmonary function following O3 exposure at rest. Symptoms of “shortness of breath,” “chest tightness,” “chest pain,” “dry, scratchy, or sore throat,” and “dyspnea” were all significantly higher from baseline measurement immediately post-exposure, and “shortness of breath,” “chest tightness,” and “dyspnea” remained greater than baseline at 30 mins post exposure. This combination of symptoms suggests activation of a lung irritant response that causes discomfort in the chest and throat, particularly upon deep inspiration, and leads to feelings of breathlessness. Although not directly measured, when examining spirometry flow-volume loops following O3 exposure, coughing was prevalent in nearly all participants, further indicating an irritant receptor response within the lungs. Large decreases in flow rates, as measured by PEFR, FEV1, and FEF25-75 were also observed immediately and 30 mins post resting exposure to 750 ppb O3. Decreases in FEV1 and PEFR are likely impacted by both decreased recoil pressure due to lower total lung capacity and potential medium-to-large airway obstruction, whereas decreased FEF25-75 represents dysfunction within the medium-to-small airways (Kim et al., 2021). These changes are likely driven by 69 reflexive responses in the airways that are designed to reduce exposure to harmful chemicals by reducing ventilation, as well as damage to the respiratory tract (ISA for Ozone and Related Photochemical Oxidants, 2020). Activation of sensory vagal afferents in the lower airways can alter the autonomic activity via the parasympathetic cholinergic pathway, causing airway obstruction and increased airway responsiveness (Lee & Widdicombe, 2001). Local changes via activation of C-fiber sensory nerves can cause axon reflex responses, contributing to further airway obstruction and hyperreactivity, while also contributing to reduced ability to maximally inspire to total lung capacity (ISA for Ozone and Related Photochemical Oxidants, 2020). Further changes due to inflammation within the airways may also occur, as inflammatory markers have been found to be present in bronchoalveolar lavage fluid as early as one hour after exposure to O3 (Koren et al., 1991). Although results appear comparable to previous work, significant challenges were encountered in obtaining proper pulmonary function tests from participants immediately following resting O3 exposure at 750 ppb. This concern was first documented by Silverman et al. in 1976, and in practice often required multiple trials beyond the minimum of three recommended by the American Thoracic Society (ATS) to achieve repeatability of FVC and FEV1 of equal or less than 0.15L. A 10-minute window was provided for participants to complete spirometry measures immediately following resting exposure, to allow for distinction between the 0-minute and 30-minute post-exposure measures. However, some participants were unable to meet repeatability requirements within the time window, or were unwilling to complete further trials due to discomfort. This is likely due to a strong irritant receptor response that made deep inspiration challenging, and caused coughing on forceful expiration. Many trials were not suitable for analysis given significant coughing during the trial causing early termination. 70 However, repeatability was much more easily achieved at the 30-minute post testing time point, as responses were slightly less strong. 4.7 Individual Susceptibility of Responses Although we observed strong changes in symptom development, pulmonary dysfunction, breathing pattern, and metabolic variables in response to O3, it is important to recognize the variability of these responses. Even within the homogenous sample of healthy, highly trained endurance athletes recruited for this thesis study, some participants had responses much greater than group means, whereas other participants did not demonstrate a response. This pattern of responses is well documented in previous literature, although little consensus as to why certain individuals respond differently than others is present. Pulmonary function responses to O3 are thought to be related largely to intrinsic responsiveness and are highly reproducible under repeated testing (Kleeberger, 1995), with responses between individuals becoming more variable with greater pollution doses (McDonnell et al., 1983). These effects are also seen with exercise performance measures, wherein some individuals experience more severe decreases than others during graded maximal exercise tests (Horvath et al., 1979) or time trial performances (Gomes et al., 2010). As a result, drawing group-wide conclusions regarding the effects of O3 exposure are challenging, given the wide range of individuality in responses. When examining the results of this thesis study, there is clearly a wide range of interindividual responsiveness to O3 exposure. However, the use of a crossover design, where each participant completed both an O3 and room air condition allows for a more robust comparison. Participants who experienced a strong O3-induced response show clear differences between experimental conditions, even in variables that did not reach significance on a group level. On the other hand, some participants exhibited no O3-induced response, and showed 71 similar results between the two experimental conditions. For example, one participant (Participant 1) demonstrated a strong change in breathing pattern during the 30-minute CWR bout, with a 21 breath/min increase in frequency, and a 1.13L/breath decrease in tidal volume in the O3 condition compared to room air. Other participants (Participant 7) experienced essentially no symptoms or pulmonary function changes with resting O3 exposure at 750 ppb, while group averages showed strong statistically significant decreases in pulmonary function and increases in respiratory symptoms. Clearly, responsiveness to O3 is highly individual, which makes the interpretation of grouped results more challenging. 5.1 Study Limitations A significant limitation of this research study is the utilization of constant workload cycling bouts, rather than allowing participants to conduct their own pacing strategy. As real-world training and competition performance is impacted by constant fluctuations in speed and effort depending on the terrain, the subject’s own perceived exertion, and their competitors, removal of this element by fixing each athlete at an individualized but constant workload diminishes the external validity of this study (Noakes, 2008). Although using fixed workloads improved the ability to assess ventilatory patterns between conditions, which are easily impacted by small changes in workload, greater clarity as to the mechanistic changes that occur under acute O3 exposure were at the expense of transferability of results to real-world athletic scenarios. Future studies with a focus on exercise performance should use a set distance time trial format, allowing athletes to control their own speed and workload throughout the task to best mimic athletic competition and training. 72 Chapter 5: Significance 5.2 Potential Impact The results of this study will add to the existing scientific literature around interindividual variability in response to O3, and how exercise capability may be impacted by exposure. We have demonstrated strong changes during maximal exercise at real-world levels of O3, at both peak exercise and during shared time points. These results assist in elucidating the complex mechanisms by which O3 exposure limits O2 transport and reduces exercise performance. With high profile athletic competitions, such as the Olympic Games, often held near metropolitan areas with high ambient levels of pollutants, understanding susceptibility to adverse effects of O3, and the corresponding mechanisms is of increased importance (Peiser & Reilly, 2004). 5.3 Future Directions This study adds to previous research examining the effects of real-world levels of O3 exposure on exercise capability in healthy athletes. Future studies in two main areas are recommended to gain further understanding of this topic area. First, in order to better understand how O3 exposure impacts oxygen delivery to the muscle, future studies focusing on mechanisms should employ techniques such as measuring alveolar ventilation, diffusion capacity, muscle oxygen saturation, and bronchoalveolar lavages. Analyzing different steps of the O2 transport cascade would allow greater certainty to the theories of the mechanisms described above. Second, in order to better understand the implications for performance changes, studies with a focus on performance should allow for participants to control gearing choices and effort during performance tests, and not employ a fixed wattage time-to-exhaustion test. Understanding the psychological decision-making processes of athletes exposed to O3 pollution, and how this 73 might impact pacing strategy and other competitive choices is of great importance to real-world competitions, which are commonly exposed to high levels of O3. 74 Chapter 6: Conclusion With increased global urbanization, an increased percentage of the population is exposed to potentially hazardous air pollutants when performing exercise. Ground-level ozone (O3) is well recognized to negatively impact pulmonary function and causes increase respiratory symptoms when inhaled, during exercise these effects are less clear. 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Thesis/Dissertation
eng
Kinesiology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Koehle, Michael
Investigating the susceptibility of cardiorespiratory responses to ozone during cycling exercise
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