Investigating Susceptibility of Cardiorespiratory Responses to Ozone During Cycling Exercise Based on Resting Ozone Exposure
Phase: NA
Conditions: Ozone Exposure During Rest and Exercise
Sponsor: University of British Columbia

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Methods 
1.1 Sample and Recruitment 
 
The target population for this study is healthy athletes with a competitive background in 
cycling or other similar endurance-based sports, such as running, swimming, or cross-country 
skiing. The predicted sample representing this population will be individuals with a background 
in competitive endurance sports living and training in the Greater Vancouver area. Recruitment 
will take place by non-probability sampling methods, mainly by purposive sampling. Copies of a 
poster will be placed on public advertising boards around the University of British Columbia 
(UBC) Vancouver campus, as well as online on the UBC School of Kinesiology website, and 
will be distributed via email 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). This poster will be 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. The poster will also be circulated on social media (Facebook, Instagram, Strava, 
Reddit, etc.) to reach a greater audience of potential participants. 
Table 1. Inclusion and exclusion criteria for the proposed research study. 
Inclusion Criteria 
Exclusion Criteria 
1) Currently training and/or competing in 
endurance sport 
2) Maximal oxygen consumption 
(VO2max) greater than 60 ml/kg/min 
(males) or 55 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 
subjects 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 suspect you may be 
pregnant 
 

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Table 1 above outlines the inclusion and exclusion criteria for the proposed research 
study. The first inclusion criterion, athletes currently training and/or competing in endurance 
sport, represents the target population for this study. As this study is designed in collaboration 
with the Canadian Sport Institute to access susceptibility to ozone exposure in national and 
international level athletes, it is logical to only recruit athletes to maximize transferability of 
results. Similarly, the second inclusion criterion, maximal oxygen consumption (VO2max) 
greater than 60 ml/kg/min (males) or 55 ml/kg/min (females), helps ensure that the sample is 
representative of the target population. Although elite endurance athletes have been shown to 
achieve VO2max values well above 70 ml/kg/min (Jones et al., 2021), such athletes are unlikely 
to participate in the proposed study to avoid interrupting their rigid training programs. The 
specified VO2max requirement will allow for well-trained individuals in roughly the ~90th 
percentile of aerobic fitness (Kaminsky et al., 2022), without the time and training constraints of 
professional athletes to participate in the proposed study. Additionally, by reducing the 
variability of fitness level between participants, it is likely that the variability between minute 
ventilation during exercise will also be reduced, helping ensure that a similar effective dose of 
pollution is delivered to all participants. The third inclusion criterion, between the ages of 18 and 
50, reduces 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 subjects over the age of 18 will be 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 will not be permitted to 

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participate due to increased risk. The fourth inclusion criterion, ability to communicate 
sufficiently in the English language, ensures that subjects are able to fully understand study 
procedures, have the ability to ask questions, and provide informed consent. 
 
The fifth inclusion criteria, taking hormonal birth control (females only), minimizes 
potential confounding factors on inflammatory responses in females due to the potential 
protective effects of estrogen (Birukova et al., 2019). By removing variation in sex hormones 
that naturally occurs 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, avoids potential biasing of expired nitric oxide 
(FeNO) measurements. It is well recognized that both acute and chronic exposure to smoking is 
associated with significant reductions in FeNO, due to increased breakdown of nitric oxide (NO) 
(Malinovschi et al., 2006). Additionally, acute rhinovirus infections are shown to increase FeNO 
50-150% due to upregulated inducible nitric oxide synthase (iNOS) in the respiratory tract 
(Bjermer et al., 2014). As such, exclusion of smokers and those not fully recovered from 
infection will best ensure FeNO measurements are indicative of lung inflammation. The third 
exclusion criteria, presence of any chronic respiratory disease, helps mitigate the influence of 
such diseases on spirometry measures. Those with respiratory diseases that cause airway 
hyperreactivity, 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 aims to 
establish susceptibility and ventilatory responses to ozone in healthy subjects, removal of 
subjects with chronic respiratory diseases will limit potentially conflicting factors. 

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The fourth exclusion criteria, current symptoms or infection with COVID-19, helps 
mitigate any potential influences of both short- and long-COVID-19 symptoms, as well as 
protecting 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 suspect you may be pregnant, minimizes risk 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 
subjects who may be pregnant increases participant safety. 
 
Sample size was calculated using data from previous studies that examined changes in 
FEV1 in athletes following ozone exposure during exercise. A similar crossover design in elite 
athletes utilizing ozone exposure at 200ppb 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 subjects. Sample size will be 
increased to 24 to include female subjects. 
1.2 Measures/Instruments 
O3 will be 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 will be 
delivered to subjects via a tube connected to the inspiratory valve on a three-way non-rebreathe 
mouthpiece. The Thermo ScientificTM 49iQ Ozone Analyzer (Franklin, MA, USA) will be 
attached to the inspiratory tube and used as an O3 sensor. Calibration will occur according to the 

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manufacturing standards using the Thermo ScientificTM 49iQ Ozone Primary Standard 
generator.  
Spirometry will be measured through the ParvoMedics OUS-SPIRO on the metabolic 
cart (Salt Lake City, UT, USA), which will be calibrated prior to each use. While wearing a 
nose-clip, participants will be asked to complete a maximal inspiration to maximal lung capacity 
followed by a maximal forceful exhalation for 6-seconds, following breathing out at the end of a 
normal breath. This will allow for measurement of forced expiratory volume in one-second 
(FEV1), forced vital capacity (FVC), forced mid-expiratory flow (FEF25-75), and peak expiratory 
flow rate (PEFR). Based on guidelines provided by the American Thoracic Society (ATS), trials 
will be repeated a minimum of 3 times until the two largest FEV1 and FVC values differ by less 
than or equal to 150mL (Graham et al., 2019). Spirometry values will be compared between the 
two experimental trials to examine how O3 exposure may impact development of expiratory flow 
limitation. 
 
Fractional nitric oxide concentration in expired breath (FeNO) will be measured by a 
calibrated 2nd generation NOBreathe® FeNO monitor (Bedfont® Scientific Ltd., England). 
Participants will be instructed to perform a deep inhalation away from the mouthpiece, and 
exhale at a consistent flow rate for 12 seconds into the mouthpiece of the device until prompted 
to stop by the monitor. Four measurements will be taken at each specified time point. FeNO will 
be compared between the two experimental trials to examine how O3 exposure may impact lung 
inflammation. 
 
Symptoms will be measured via a custom brief questionnaire previously used in the 
laboratory. Before and after each exercise bout, participants will be asked to rate symptoms 
related to their eyes, nose, throat, chest, and general health on a 0-5 scale, where 0 means “you 

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don’t experience this symptom at all” and 5 means “very severe”. Symptoms will be compared 
between the two experimental trials to examine how O3 exposure may impact symptom 
development. 
Dyspnea during rest and exercise will be 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 represent 
“the most severe breathing discomfort that you have ever experienced or could imagine 
experiencing.” Ratings of dyspnea will be compared between the two experimental trials to 
examine how O3 exposure may impact dyspnea. 
Rating of perceived exertion (RPE) during exercise will be measured using the Borg 
scale (Borg, 1982). Participants will be intermittently asked to express how hard they feel like 
they are 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. 
Blood lactate will be measured via testing strips and a portable LactatEDGE analyzer 
(Warsaw, Poland) by sampling at the finger tip of the index or middle finger. The skin surface 
will be 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 will be analyzed for 
concentration of blood lactate (mmol/L). The discarded lancet will be placed in a designated 
sharps bin, while any other materials exposed to blood (gauze, alcohol swab, discarded strips) 
will be placed in the marked biohazard bin. A trained researcher wearing gloves will obtain all 
samples. Blood lactate values will be utilized to determine aerobic and anaerobic thresholds 

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during the step-incremental cycling test, as well as compared between the two experimental trials 
to examine how O3 exposure may impact production of lactate. 
Inspiratory capacity maneuvers will be performed during rest and exercise to assess 
ventilatory patterns. While connected to the mouthpiece, participants will be asked to fill up their 
lungs completely from the end of a normal expiration. The volume of air inspired during these 
tasks will represent the inspiratory capacity of the subject, and will be compared between the two 
experimental trials to examine how O3 exposure may impact dynamic hyperinflation of the lungs 
during exercise. 
1.3 Study Design 
 
This research design can be characterized as a double-blind crossover with pre- and post- 
measures. Ozone pollution status is the independent variable, of which there are two levels 
during the experimental trials: ozone exposure at 170 parts per billion (ppb) O3, and room air 
(<10 ppb O3). Testing will take place over four laboratory sessions, with a minimum of 72 hours 
of washout between each visit. During the first session, subjects will undergo a step-incremental 
test on a cycle ergometer to determine subject characteristics and identify anaerobic threshold, 
followed by familiarization of exercise pulmonary function tests. During the second visit, 
subjects will undergo resting spirometry and assessment of exhaled nitric oxide before and after 
exposure to 120 minutes of O3 at 750ppb to characterize resting response. The third and fourth 
laboratory visits will consist of 30-minutes of cycling at the workload associated with 10% 
below previously established anaerobic threshold with exposure to either ozone or room air. 
Post-exercise spirometry and FeNO measures will be obtained prior to a time to exhaustion test 
at 105% of previously established peak power output. During the O3 condition, subjects will be 
exposed to 170ppb for 60 minutes prior to, and during the cycling tasks, whereas during the 

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control condition subjects will breathe room air for the identical procedures. Both the primary 
researcher and subjects will be blinded to the experimental condition for each visit, with the 
order randomized for each subject. 
1.4 Procedures 
 
Participants will visit the laboratory for testing on 4 occasions. A minimum of 72-hours 
washout is required between visits. Before each session involving exercise (Visits 1,3,4), 
subjects will be 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 will include, but is not limited to, avoiding alcohol and 
strenuous exercise for 24 hours, ensuring a proper sleep the night prior, and consuming a pre-
exercise meal that they are accustomed to timed appropriately prior to each visit. However, 
participants will be 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 utilize such strategies during competition. All visits involving exercise (Visits 1,3,4) 
will be performed at a similar time of day in order to avoid any diurnal variation in exercise 
performance (Kusumoto et al., 2021). 
 
The first visit will involve participant consent, screening for inclusion based on a 
maximal exercise test, and familiarization with pulmonary function measurements. Participants 
will first re-read the consent form. They will be given the opportunity to ask any questions 
related to their involvement, then be asked to sign the informed consent. Participants will also 
complete a PAR-Q+ form, which will be reviewed prior to partaking any exercise in the 
laboratory. Participants will then be familiarized with pulmonary function testing equipment and 
protocols, and undergo resting spirometry and FeNO measurements (described in detail above). 

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Spirometry will involve maximal inspiration followed by maximal forceful expiration to 
determine FEV1, FVC, FEF25-75, & PEFR. FeNO will be measured by expiring into the portable 
device 4 times to assess lung inflammation. Following resting pulmonary function 
measurements, subjects will complete a 10-minute warmup at 1-watt/kilogram body weight on a 
Velotron DynaFit Pro cycle ergometer, before performing a step-incremental graded exercise test 
(GXT). The step-incremental GXT will consist of 3-minute stages, starting at 100 watts with 
power output increasing by 25 watts/stage, until the subject reaches volitional exhaustion, is 
unable to maintain 60 revolutions per minute (RPM), or is unwilling to continue. Blood lactate 
will be sampled via finger prick during the last 30-seconds of each stage, and subjects will be 
asked to rate their perceived exertion at the end of each stage. Following completion of the test, 
subjects will be given a brief recovery before cycling for 5-minutes at the power output 10% 
below their previously calculated anaerobic threshold to mimic experimental visits 3 and 4. 
Anaerobic threshold will measured as the work rate associated with blood lactate at 4mmol/L, 
indicating a change from metabolic steady state to a non-steady state attainment of VO2max. 
During this constant workload bout, subjects will be instructed as to how to complete an 
inspiratory capacity maneuver, provide rating of perceived exertion, and rate their feelings of 
dyspnea. Subjects will then perform post-exercise pulmonary function measures identical to that 
completed at rest, prior to completing a verification exercise bout (VEB) at 105% of the 
previously achieved maximum work rate. Subjects will be asked to cycle for as long as possible 
at the specified work rate until they are no longer able to maintain 60 RPM, and time-to-
exhaustion will be recorded. Subjects will then perform a final round of pulmonary function 
measures. Participants will be suited with a mouthpiece connected to a three-way non-rebreathe 

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valve, and expired gas and flow will be analyzed by a ParvoMedics’ TrueOne 2400 metabolic 
measurement system (Sandy, UT, USA).  
 
The second visit will consist of resting exposure to ozone and pulmonary function tests. 
Subjects will conduct pre-ozone resting spirometry, FeNO, dyspnea, and symptom 
measurements, followed by 2-hours of resting O3 exposure at 750ppb. During the resting 
exposure, subjects will be seated with a three-way non-rebreathe valve and mouthpiece 
connected via tube to the ozone chamber. Nose-clips will be worn for the entirety of the protocol, 
and expired volumes will be analyzed to determine average minute ventilation, and effective 
dose of O3 (minute ventilation x O3 concentration x exposure duration) (Silverman et al., 1976). 
Subjects will be permitted to use personal electronic devices (phone, laptop, tablet etc.) during 
exposure, provided no significant movement is required. Following exposure, subjects will 
complete identical spirometry, FeNO, symptoms, and dyspnea measurements immediately and 
30-minutes post-exposure to that completed pre-exposure. Post-exposure and pre-exposure 
values will be compared to determine resting response to O3.  
 
The third and fourth visits will consist of identical procedures, randomized as to the order 
of the O3 and control conditions. During both experimental trials, a lab member other than the 
researcher will control the O3 concentration in order to maintain blinding of the participant and 
the researcher as to the condition. During the O3 exposure, subjects will receive 170ppb O3, and 
during the control exposure subjects will receive room air (typically <10ppb O3). Upon arrival in 
the laboratory, the researcher will confirm that pre-visit requirements (detailed above) are met, 
prior to resting spirometry, FeNO, symptoms, and dyspnea measurements. Subjects will then be 
connected via tube and mouthpiece/valve to the O3 chamber. While remaining connected to the 
O3 chamber, participants will then complete a standardized warmup on the Velotron of 25-

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minutes at 10% below previously established aerobic threshold. Subjects will then be provided 
with 5-minutes of rest prior to completing 30-minutes at 10% below anaerobic threshold. 
Expired gas and volumes will be analyzed throughout the trial, and participants will perform an 
inspiratory capacity test, have blood lactate sampled via finger-prick, rate their perceived 
exertion and dyspnea every 10-minutes (10,20,30-minutes into exercise). Post-exercise, subjects 
will remove their mouthpiece and complete spirometry, FeNO, dyspnea, and symptoms testing 
(~5mins), prior to reattaching the mouthpiece and completing a time-to-exhaustion test at 105% 
of peak power achieved during the GXT. Strong verbal encouragement will be provided to 
subjects, and exercise will be stopped when they are no longer able to maintain 60 RPM, and 
time-to-exhaustion will be recorded. Post-exercise, a final set of pulmonary function, symptoms, 
and dyspnea testing will occur at 30-minutes following completion of the submaximal exercise 
bout. 
1.5 Data Analysis 
 
As the purpose of this research study is to assess the interaction between O3 exposure and 
cardiorespiratory function during rest and exercise in healthy athletes, we aim to characterise the 
relationship between resting response and exercise response, as well as assess the effects of 
ozone exposure on pulmonary function, ventilatory patterns and exercise parameters. To assess 
susceptibility to exercising O3 response, a regression analysis will occur between the reduction in 
spirometry values post 2-hours resting exposure at 750ppb and the reduction in spirometry 
values post-O3 condition exercise trial. It is hypothesized that greater decrements in FEV1, FVC, 
FEF25-75 and PEFR will occur following exercise under O3 exposure in subjects who also 
exhibited greater expiratory flow limitations following resting exposure. 

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Examining ventilatory patterns during exercise is another primary objective of the 
proposed research study. By comparing tidal volume, breathing frequency, and inspiratory 
capacity throughout the constant work-rate exercise bout between the two experimental 
conditions through mixed models, further insight into how O3 exposure may impact ventilation 
may be gained. 
 
Analyzing time to exhaustion (in seconds) for the maximal constant work-rate exercise 
bouts between the two experimental conditions will also be assessed by mixed models. Potential 
differences between the two conditions will help elucidate whether performance changes occur 
due to O3 exposure. Further analysis of ratings of perceived exertion, dyspnea, and FeNO via 
mixed models will also occur between the two experimental trials.