Understanding and Managing the Limits of Physiological Tolerance in Heat Vulnerable Canadians During Rest and Physical Exercise
Phase: NA
Conditions: Aging, Heat Stress, Physiological Stress
Sponsor: University of Ottawa

 
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STUDY PROTOCOL AND STATISTICAL ANALYSIS PLAN 
 
 
Project: Physiological Responses in Young and Older Adults During a 
Prolonged Simulated Heatwave (H 05-16-07)  
 
 
Prepared by: Robert D. Meade, PhD1,2, Ashley P. Akerman, PhD1,3, and 
Glen P. Kenny, PhD1,4 
 
1Human and Environmental Physiology Research Unit, School of Human Kinetics, 
University of Ottawa, Ottawa, Ontario, Canada,  
 
2Harvard T.H. Chan School of Public Health, Harvard University, Boston, MA, USA. 
 
3Ultromics Ltd, Oxford, UK 
 
4Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, Ontario, 
Canada.   
 
 
NCT number: NCT04353076 
 
Date uploaded: August 11th, 2021 
 
 
Address for correspondence: 
Dr. Glen P Kenny 
University of Ottawa, 
School of Human Kinetics, 
125 University, Room 367 
Montpetit Hall 
Ottawa, Ontario, Canada 
K1N 6N5 
(613) 562-5800 ext. 4282 
(613) 562-5149 (fax) 
email: gkenny@uottawa.ca 
 

 
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Update note 
This document provides an update of the study protocol and statistical analysis plan 
originally submitted April 13th, 2020. The employed methods and measurements, 
including the addition of measurements of heart rate variability and acute circulating and 
intracellular inflammatory and stress markers, have been described in greater detail. The 
statistical analysis plan has been refined considerably.  
Data collection was officially completed April 2nd, 2021. Outcome data was blinded on 
May 19th, 2021, and data cleaning and analysis was completed on August 11th, 2021. The 
final data set is still blinded at the time of this update.  
Note that, for the sake of readability, the grammatical tense of the document has been 
kept consistent despite the completion of data collection. 
Relevance of the research project 
Older adults are among the most at risk of acute injury during extreme heat events due, 
in large part, to impaired homeostatic responses to heat stress. However, much of our 
understanding of the effects of extreme heat on physiological responses in older adults 
has come from studies that have employed short-term (<3 hours) exposure to conditions 
hotter than those typically experienced during heatwaves. Additionally, there is a dearth 
of information regarding the physiological impacts of currently recommended heat-
mitigation strategies. For instance, the World Health Organization recommends that when 
home-cooling is unavailable, individuals should visit a cooled location (e.g., a cooling 
centre) for 1-2 hours in order to cool the body though direct evidence to support this 
guidance is lacking. Whether this intervention can provide lasting reductions in 
physiological strain is not clear, but it is likely that reductions in body temperature are only 
transient as heat will be rapidly gained by the body upon re-exposure to elevated ambient 
temperatures.  
The proposed research will provide important and novel data on i) the effect of prolonged 
exposure to simulated heatwave conditions on whole-body heat storage and resultant 

 
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thermoregulatory, cardiovascular, and inflammatory responses in young and older adults, 
and ii) the efficacy of a cooling centre intervention for limiting physiological strain.  
Research Project Objectives 
The objectives of this project are to: i) evaluate the effect of age on whole-body heat 
storage, body core temperature, and the development of cardiovascular strain and acute 
inflammation during day-long (9 hours) exposure to simulated heatwave conditions 
(Intervention 1); and ii) determine whether short-duration exposure (2 hours) to an air-
conditioned environment following extreme heat exposure results in lasting reductions in 
physiological strain in older adults upon return to the heat (Intervention 2). 
The first objective (Intervention 1) will be achieved by comparing whole-body heat 
storage (measured using combined direct and indirect calorimetry) and the accompanying 
changes in body temperature (i.e., rectal, mean skin temperature), cardiovascular, and 
circulating and intracellular inflammatory and stress responses between young and older 
adults during a 9-hour resting exposure to ~40°C and ~10-15% relative humidity 
conditions (heat index: 37-38°C). These conditions were chosen to simulate peak 
temperatures experienced during heatwaves and are similar to conditions in recent 
heatwaves in North American in 2018 (Ottawa, Ontario; 34°C and 58%, heat index: 41°C) 
and Europe in 2003 (Paris, France; 38°C and 25%, heat index: 38°C). This intervention 
will thereby allow for the determination of whether ecologically valid heatwave conditions 
exceed the physiological capacity for heat dissipation in older adults, and how thermal 
and cardiovascular strain and inflammation develops during long-duration heat exposure.   
The second objective (Intervention 2) will be accomplished by comparing physiological 
responses in the older adults in Intervention 1 to a separate age-matched group removed 
from the heat to spend 2 hours in a cooler environment (23°C, ~50% relative humidity) 
approximately mid-way (hours 5-6) through the simulated heatwave. Comparing whole-
body heat storage and body temperature, cardiovascular, and inflammatory responses 
during and after the ambient cooling intervention to the non-cooled group will allow for 
determination of whether short-term cooling confers prolonged reductions in physiological 
strain or if the cooling-induced physiological alterations occur only transiently.  

 
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Hypotheses 
Intervention 1: Age-related attenuation of the human heat loss responses (cutaneous 
vasodilation and sweating) means that older adults experience greater elevations in body 
heat storage and body core temperature compared to younger individuals during heat 
stress. For example, Kenny et al. (Temperature (Austin). 2016 31;4(1):79-88). observed 
~80% greater heat storage (equivalent to a ~0.5°C greater elevation in mean body 
temperature) in middle-aged-to-older compared to young adults during 3-hours of resting 
heat exposure. More importantly, the middle-aged-to-older adults did not achieve a state 
of heat balance (i.e., they were still storing heat within their body at the end of the 3 hours).  
Based on this work, our primary hypothesis is that older adults will not achieve a state of 
heat balance (i.e., rate of heat loss balancing the rate of heat gain; thermal equilibrium) 
and therefore experience greater heat storage throughout the 9-hour simulated heatwave 
compared to the younger participants. Consequently, body core temperature will be 
greater in the older adults and between-group differences will be exacerbated as 
exposure progresses. We will also explore the secondary hypothesis that differences in 
body temperature will be paralleled by greater alterations in cardiovascular variables and 
circulatory and intracellular markers of acute inflammation and stress in the older adults. 
Intervention 2:  Our primary hypothesis is that body heat storage will be exacerbated in 
the older adults exposed to the cooling centre intervention upon return to the heat (hours 
5-6) compared to the older adults from Intervention 1 who remained in the heat. 
Consequently, body core temperature will be comparable (statistically equivalent) 
between groups by the end of exposure (i.e., 9 hours, 3 hours after return to the heat). 
This is because body temperature is regulated through a negative feedback mechanism. 
Elevations in core and skin temperatures elicit proportional activation of cutaneous 
vasodilation and sweating to facilitate heat loss and stabilize body temperature. Thus, 
core and skin cooling will suppress heat loss and, upon return to a hot environment, heat 
will be rapidly gained until body temperature rises to an extent sufficient to activate heat 
loss and re-attain thermal equilibrium. We will also evaluate the secondary hypothesis 

 
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that, in parallel to body core temperature, any effect of cooling on cardiovascular 
responses and acute inflammation will be abated by the end of the heat event. 
METHODS 
Participants 
A total of 20 young (age: 18-30 years) and 40 older (age: 64-80 years) adults will be 
recruited for the project. Young (n=20) and older (n=20) adults will be recruited for 
Intervention 1 and a separate cohort of older adults (n=20) for Intervention 2. Details 
on the determination of the sample sizes for each intervention are provided below.  
Written and informed consent will be obtained from all volunteers prior to participation. 
Recruited participants will be homogenous for anthropomorphic characteristics and 
habitual physical activity levels as verified via standardized questionnaires and each 
intervention will have an approximately even distribution of males and females. Older 
participants will be screened for cardiovascular risk factors prior to the heatwave 
simulations.  No participants will be current smokers. The specific inclusion and exclusion 
criteria for the study are provided below 
Inclusion Criteria 
• 
Male or female (non-pregnant) adults with or without a) chronic hypertension (elevated 
resting blood pressure; as defined by Heart and Stroke Canada and Hypertension 
Canada), b) type 2 diabetes as defined by Diabetes Canada, with at least 5 years 
having elapsed since time of diagnosis and or c) obesity as defined by the World 
Health Organization (Body Mass Index [BMI] greater than or equal to 30). 
• 
Non-smoking. 
Exclusion Criteria 
• 
Episode(s) of severe hypoglycemia (requiring the assistance of another person) within 
the previous year, or inability to sense hypoglycemia (hypoglycemia unawareness). 
• 
Serious complications related to diabetes (gastroparesis, renal disease, uncontrolled 
hypertension, severe autonomic neuropathy). 
• 
Uncontrolled hypertension – Arterial blood pressure >150 mmHg systolic or >95 
mmHg diastolic in a sitting position. 

 
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• 
Restrictions in physical activity due to disease (e.g. intermittent claudication, renal 
impairment, active proliferative retinopathy, unstable cardiac or pulmonary disease, 
disabling stroke, severe arthritis, etc.). 
• 
Use of or changes in medication judged by the patient or investigators to make 
participation in this study inadvisable. 
• 
Cardiac abnormalities identified in your physical health screening forms (adults <65 
years of age and older; adults <60 years of age and older with diabetes and or 
hypertension) or during exercise stress testing as assessed by 12-lead (all adults ≥65 
years of age and older). 
Sample size calculations 
Intervention 1: An a priori power analysis determined that a total sample size of 19 young 
and 19 older adults is required to detect a difference in the rate of whole-body heat 
storage between groups at the end of each calorimeter session (i.e., hours 3 and 9) with 
80% statistical power. In lieu of clinically meaningful data (i.e., what would be considered 
a clinically meaningful change in whole-body heat storage), the standardized effect size 
(Cohen’s d = 1.06) was calculated from the difference in the rate of body heat storage 
between young and older adults over the final 30-min of a 3-hour heat exposure (young: 
-2 [26] kJ/hour, older: 43 [54] kJ/hour) in our previous work (Kenny et al. Temperature 
(Austin). 2016 31;4(1):79-88). 
Intervention 2: An a priori power analysis determined that a total sample size of 18 older 
adults in each group is required to confirm whether between-group differences in rectal 
temperature are within upper and lower bounds of +0.3°C and -0.3°C, respectively, with 
80% power. This corresponds to an effect size (Cohen’s d) of 1.0, based on the pooled-
standard deviation of 0.3°C. Equivalence bounds were chosen based on the typical day-
to-day variation of body core temperature (Consolazio  et al. Physiological measurements 
of metabolic functions in man. 1963:453-480) and suggested meaningful/detectable 
change in body core temperature in a recent study assessing the influence of personal 
cooling strategies on physiological strain in young adults (Morris et al. Annals of Internal 
Medicine. 2019;171(9):675-677). The standard deviation for the between group 
difference was determined from data from our laboratory demonstrating a 0.2°C (SD 0.3) 
difference in rectal temperature between young and older adults (Kenny et al. 

 
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Temperature (Austin). 2016;4(1):79-88) and a 0.0°C (SD 0.3) difference between older 
adults with and without type 2 diabetes (Poirier et al. Temperature (Austin). 
2020;7(3):263-269) during 3 hours of rest in the heat (44°C, 30% relative humidity).  
Experimental Design 
Pre-trial instructions  
Participants will be asked to avoid strenuous physical activity and alcohol for 24 hours 
prior to all preliminary and experimental sessions and to eat a light meal 2 hours before 
the start of each session. Participants will also be asked to consume a minimum of 500 
ml of water the night before and morning of each session to ensure adequate hydration. 
Adequate hydration will be verified upon arrival to the laboratory on the day of the heat 
event simulation via urine specific gravity (operationally defined as a urine specific gravity 
<1.025; Kenefick & Cheuvront. Hydration. Nutr Rev. 2012;70 Suppl 2:S137-142). In the 
event that participants exceed this threshold, ~500 mL of tap water will be provided, and 
urine specific gravity will be tested again after ~30 min. For all sessions, clothing will be 
standardized to shorts and sandals (and a summer top for women).  
Preliminary screening 
All participants will complete one preliminary evaluation a minimum of 7 days before the 
experimental session. During this session they will be familiarized with all procedures and 
measurements and complete the Canadian Society for Exercise Physiology Get Active 
Questionnaire (GAQ) and the American Heart Association Pre-Participation Screening 
Questionnaire to assess their eligibility to participate. The GAQ will also be used to assess 
habitual activity levels along with the Kohl Physical Activity Questionnaire to determine 
the general types of physical activity performed. Arterial blood pressures will then be 
evaluated in triplicate via manual auscultation (~30 sec between measures). Thereafter, 
participant physical characteristics will be evaluated. Body height and mass will be 
determined via a physician stadiometer (Detecto, model 2391, Webb City, MO, USA) and 
a high-performance weighing terminal (model CBU150X, Mettler Toledo Inc., 
Mississauga, ON, Canada), respectively. Body mass index and surface area will be 
calculated from these measures.  

 
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Intervention 1: Physiological responses in young and older adults exposed to a simulated 
extreme heat event 
Each experimental session will commence between 07:00-09:00. Upon arrival to the 
laboratory, the participant will provide a urine sample for the assessment of urine specific 
gravity and insert a temperature probe for the measurement of rectal temperature (Mon-
a-therm General Purpose Temperature Probe, Mallinckrodt Medical Inc.). A nude body 
mass will then be obtained, and the participant will be instrumented with a 5-lead 
electrocardiogram (Philips DigiTrak XT Holter Monitor, Philips) and skin temperature 
sensors (DS1922L Thermochron, OnSolution Pty Ltd) at 8 body regions: forehead, right 
scapula, upper left chest, upper right arm, right forearm, left hand, right anterior thigh, and 
left calf.  
Baseline cardiovascular parameters will then be evaluated via a brief (~45 min) 
cardiovascular test battery, performed as follows. Brachial arterial systolic and diastolic 
pressures reconstructed from arterial pressure waveforms measured at the right middle 
finger using the volume clamp technique (Finometer Pro, Fina-press Medical Systems) 
and 5-lead echocardiogram recordings (Philips DigiTrak XT Holter Monitor, Philips)  will 
be collected for 10-min while the participant rests quietly (spontaneous breathing). 
Immediately thereafter, arterial systolic and diastolic pressures will be measured in 
triplicate via manual auscultation (~30 sec between measures), after which forearm and 
calf blood flows on the right side of the body will be measured via automated venous 
occlusion plethysmography (Hokanson AI6, D.E. Hokanson, Inc.). Throughout the short 
cardiovascular test battery, the participant will be seated with both feet on the floor, except 
for during the measurements of limb blood flow, where the instrumented limbs will be 
elevated to facilitate venous drainage. Finally, a venous blood sample and body mass 
measurement will be obtained.  
The participant will then be transferred to the whole-body air calorimeter chamber (a 
unique device that allows for direct quantification of heat gain/loss by the body), housed 
within a thermal chamber regulated to ~40°C and ~10-15% humidity (heat index: 37-
38°C). These conditions were chosen to simulate peak temperatures experienced during 
heatwaves and are similar to peak conditions in recent heatwaves in North American in 

 
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2018 (Ottawa, Ontario; 34°C and 58%, heat index: 41°C) and Europe in 2003 (Paris, 
France; 38°C and 25%, heat index: 38°C). The participant will sit quietly for 3-hours (hours 
1-3) within the calorimeter chamber while whole-body heat exchange is measured. 
Metabolic heat production will be evaluated simultaneously via indirect calorimetry 
(AMETEK model S-3A/1 and CD 3A, Applied Electrochemistry, Bastrop, TX, USA). At the 
3-hour mark, participants will exit the calorimeter, and the brief cardiovascular test battery 
will be performed again, followed by a measurement of body mass.  
Hours 4-6 will be spent resting in the heat in the thermal chamber (but outside of the 
calorimeter). During this time, the participant will be allowed to consume a light (~300 g), 
self-provided lunch with low water content (e.g., peanut butter sandwich). Tap water will 
be available ad libitum via a self-service insulated water cooler located in the thermal 
chamber (out of direct sight). Another cardiovascular battery will be performed followed 
by a measurement of body mass at the end of this period. The participant will then be 
transferred to the calorimeter where the final 3 hours will be spent resting in the seated 
position (hours 6-9). At the 9-hour time point, the participant will be removed from the 
calorimeter, the cardiovascular test battery was conducted, and a venous blood sample 
and body mass measurement will be procured. The participant will then be provided with 
water and/or a commercially available sports drink before leaving the laboratory.  
Intervention 2: Efficacy of a cooling centre intervention for limiting physiological strain in 
the elderly  
The protocol for Intervention 2 will be identical to that of Intervention 1 except that after 
the first calorimeter session and the subsequent cardiovascular battery, the participant 
will be removed from the thermal chamber to spend 2 hours (hours 5-6) seated in an air-
conditioned room (~23°C, ~50% relative humidity). Like protocol 1, the participant will be 
able to consume water (tap) ad libitum and eat a small self-provided during this time. The 
third cardiovascular battery will be performed in the cooled environment. At the end of the 
6th hour, participants will re-enter the calorimeter where they will spend the remaining 3 
hours of exposure seated in the heat.  
Measurements 

 
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Primary Outcome measures 
Whole-body heat storage and body core temperature 
The primary aim of the study is to assess differences in body temperature regulation 
between young and older adults (Intervention 1) and the efficacy of a cooling centre 
intervention for limiting hyperthermia in the elderly (Intervention 2) during a 9-hour 
simulated extreme heat event. Body temperature regulation will be quantified as between-
group differences in whole-body cumulative heat storage (i.e., the total amount of heat 
stored in the body) and body core temperature (rectal temperature, surrogate measure of 
body heat content) in each intervention. 
Whole-body evaporative and dry heat loss will be continuously measured during the first 
and final 3 hours of exposure via the Snellen air calorimeter, which provides the only 
standard measure of these metrics. Calorimeter inflow and outflow values of absolute 
humidity and air temperature will be measured at 8 second intervals using high precision 
dew point hygrometry (Model 373H, RH Systems) and resistance temperature detectors 
(Black Stack model 1560, Hart Electronics), respectively. Air mass flow, equivalent to 
<0.3 m/s where the participant is seated, will be determined via differential thermometry 
over a known heat source in the effluent air stream. All data will be displayed and recorded 
on a personal computer with LabVIEW software (Version 7.0, National Instruments). Heat 
loss via sweat evaporation will be then determined using the outﬂow–inﬂow difference in 
absolute humidity, multiplied by air mass ﬂow and the latent heat of vaporization of sweat 
(2427 J/g). Dry heat loss will be similarly derived from the outﬂow–inﬂow air temperature 
difference and speciﬁc heat capacity of air (1005 J/kg/°C). Ambient temperature (40°C) 
will be greater than that of the skin (~35-36°C), meaning that a negative dry heat loss will 
be measured. Dry heat exchange was therefore expressed as a dry heat gain. 
Metabolic energy expenditure will be determined using indirect calorimetry. Expired 
oxygen (O2) and carbon dioxide (CO2) content will be measured with electrochemical gas 
analysers (AMETEK model S-3A/1 and CD 3A, Applied Electrochemistry) from air drawn 
from a 6 L fluted mixing box located within the calorimeter. Expelled air will be recycled 
back into the chamber to account for respiratory heat exchange. The gas analyzers and 

 
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turbine ventilometer will be calibrated ~30 min prior to each 3-hour calorimetry 
measurement period. Endogenous metabolic heat production will be assumed to be 
equivalent to metabolic energy expenditure since no external work will be performed. 
Cumulative body heat storage will be evaluated as the temporal summation of total heat 
gain (heat production + dry heat gain) and evaporative heat loss.  
Rectal temperature will be monitored using a general-purpose thermocouple temperature 
probe (Mon-a-therm General Purpose Temperature Probe, Mallinckrodt Medical Inc.) 
inserted ~12 cm past the anal sphincter with data collected in 15-s intervals using 
LabVIEW software (Version 7.0, National Instruments).  
Secondary Measures 
The secondary aim of the project is to evaluate the extent to which age-related alterations 
in body temperature regulation (Intervention 1) and short-term ambient cooling 
(Intervention 2) are associated with alterations in physiological responses that can 
contribute to dysfunction and injury during a prolonged extreme heat event (Meade et al. 
Environ Int. 2020;144:105909). These include whole-body mean skin temperature and 
clinically relevant cardiovascular responses, as well as circulating and intracellular 
markers of inflammation and stress. 
Mean skin temperature 
Skin temperature will be assessed every minute using surface temperature monitors 
(DS1922L Thermochron, OnSolution Pty Ltd) affixed to 8 body regions, using double-
sided adhesives and medical tape, as described in ISO 9886:2004. Mean skin 
temperature will then be calculated using the provided weightings: forehead (7%), right 
scapula (17.5%), upper left chest (17.5%), upper right arm (7%), right forearm (7%), left 
hand (5%), right anterior thigh (19%) and left calf (20%). 
Cardiovascular measurements  
Electrocardiogram data from Holter monitor (Philips DigiTrak XT Holter Monitor, Philips) 
will be downloaded and analysed using Philips Zymed Software (Philips Zymed Version 
3.0, Andover, MA). R-R interval data will be extracted from the ECG tracing (3 channel at 

 
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a sampling rate of 175 Hz). Normal-to-normal beats, as determined by the Zymed 
annotation algorithm, will be retained for further analysis. Continuous Individualized 
Variability 
Analysis 
(CIMVA) 
software 
(http://ohridal.org/cimva/CIMVA-
CoreDescription.pdf) will be used to derive absolute heart rate and indices of heart rate 
variability (5 min windowed analysis with 30 sec time step). The latter includes 1) standard 
deviation of successive normal-to-normal intervals (SDNN), an index of overall variability; 
and 2) the root mean squared of the standard deviation (RMSSD) of successive normal-
to-normal intervals, which is more reflective of short-term high frequency fluctuations in 
heart rate (mediated primarily by the parasympathetic nervous system).  
Arterial systolic and diastolic blood pressures will be intermittently taken as an average 
of the three values measured at the brachial artery (~30 s between measures) via manual 
auscultation. Mean arterial pressure will be calculated as 2/3 diastolic pressure + 1/3 
systolic pressure. Rate pressure product, an index of myocardial work, will also be derived 
as heart rate × systolic pressure.  
Brachial arterial blood pressures and cardiac output will be estimated from beat-to-beat 
recordings of arterial pressure waveform measured at the right middle-finger using the 
volume-clamp technique (Finometer Pro, Fina-press Medical Systems). Stroke volume 
will be calculated as cardiac output ÷ heart rate, whilst total peripheral resistance is 
derived as mean arterial pressure ÷ cardiac output. Cardiac baroreflex sensitivity will be 
determined via the sequence technique using software provided by Finapres Medical 
Systems (PRVBRS, Fina-press Medical Systems). Forearm and calf blood flows will be 
determined as the average of a minimum of four measurements obtained via automated 
venous occlusion plethysmography (Hokanson AI6, D.E. Hokanson, Inc.). 
Circulating and intracellular inflammation and stress markers 
Venous blood will be collected for the determination of circulating and intracellular 
inflammation/stress responses. Samples collected from the antecubital vein at baseline 
and the end of exposure will be transferred directly into Vacutainer tubes with no additive 
(1 x 5 mL SST™ Serum Separator Tube, BD) or potassium ethylenediaminetetraacetic 
acid (1 x 3 mL and 3 x 10 mL K2EDTA [7.2 mg and 18 mg, respectively], BD).  

 
13 
Non-additive blood will be let to sit for 20 min to clot before centrifugation at 1.38 relative 
centrifugal force (RCF) for 10 min (room temperature). Separated serum will then be 
transferred into polypropylene Eppendorf tubes, and frozen and stored at -80ºC. Serum 
concentrations of tumor necrosis factor alpha (TNF-α, DY210-05, Bio-Techne), 
interleukin-6 (IL-6, DY206-05, Bio-Techne) and C-reactive protein (CRP, DY1707, Bio-
Techne) will be analysed via an enzyme-linked immunosorbent assay (ELISA) kits with 
provided ancillary reagents (DuoSet ELISA Ancillary Reagent Kit 2, DY008, Bio-Techne) 
according to the manufacturer’s protocol. A 1:2 sample to reagent diluent ratio will be 
used, and plates will be read on a plate reader at a wavelength of 450 nm (Synergy, 
Biotek). Circulating protein concentrations will be corrected for the change in plasma 
volume during heat exposure (described below). 
Whole blood (from K2EDTA tubes) will be immediately layered on histopaque 
(Histopaque-1077, Sigma-Aldrich) and centrifuged at 0.8 RCF for 30 min (22°C). The 
peripheral blood mononuclear cell (PBMC) layer will then be separated and washed with 
phosphate buffered saline (P4417, Sigma-Aldrich) and centrifuged again at 1.1 RCF for 
10 min (4°C). Isolated PBMCs will be resuspended in phosphate buffered saline, 
transferred to 1.5 mL microcentrifuge tubes, and centrifuged a final time at 9.8 RCF for 
10 min (4°C). Any remaining supernatant will be aspirated prior to storing the PBMC pellet 
at -80°C until analysis.   
For analysis, the PBMC pellet will be lysed on ice for 30 min in a lysis buffer consisting of 
Tris-HCL 8.0 pH (15568-025, Invitrogen, Thermo Fisher Scientific), 0.5M EDTA (15575-
020, Invitrogen, Thermo Fisher Scientific), 1.5M NaCl (S3014, Sigma-Aldrich), 1% Triton 
X 100 (X100, Sigma-Aldrich), and freshly added protease (78430, Thermo Fisher 
Scientific) and phosphatase (7842, Thermo Fisher Scientific) inhibitors. The lysate is then 
quantified (DC-protein assay, 5000112, Bio-Rad) and normalized to a total protein 
concentration of 40 µg, which was then electrophoresed on 12% polyacrylamide SDS-
PAGE gels.  
The proteins are then electrotransferred to nitrocellulose membranes (162-0112, Bio-
Rad) then blocked in tris buffered saline (TBST) (150 mM NaCl, pH 8.0) containing 0.2% 
polysorbate detergent (Tween 20; 170-6531 Bio-Rad) and 5% powdered milk (170-6404, 

 
14 
Bio-Rad) for 60 min at 4°C with gentle agitation. After blocking, nitrocellulose membranes 
will be incubated at 4°C over-night with primary antibodies (HSP70-inducible form 1:1000, 
no. 4876S, Cell Signaling Technology; HSP90, Cell Signaling Technology, no. 4875S; 
TNF-α P300A Invitrogen, Thermo Fisher Scientific; IL-6, PA1-26811, Invitrogen, Thermo 
Fisher Scientific; β-actin HRP conjugate 1:10000, no. 12262S, Cell Signaling 
Technology). Following primary antibody incubation, the membranes will be washed 
repeatedly (3 times, 10 min each) in TBST and incubated with an HRP-conjugated 
secondary antibody (Anti-rabbit IgG, HRP-linked, no. 7074, Cell Signaling Technology) at 
room temperature for 1 hour and then washed again 3 times in TBST  (10 min per wash). 
The nitrocellulose membranes will be visualized using the Clarity Max Western ECL 
Blotting substrate (1705062, Bio-Rad) and C-Digit Blot Scanner (LI-COR). Membranes 
will be stripped and re-probed with Restore Western Blot Stripping Buffer according to the 
manufacturer instructions (no. 21063, Thermo Fisher Scientific). Protein bands will be 
quantified by Image Studio Digits software (LI-COR, version 5.2) and normalized to β-
actin, which served as an internal control.  
Hydration-related variables 
Baseline (start of session) urine specific gravity will be assessed with a hand-held total-
solids refractometer. (Reichert TS 400 total solids refractometer, Reichert). Participants 
presenting to the laboratory with urine specific gravity <1.025 will be considered 
adequately hydrated to begin the experimental session (Kenefick & Cheuvront. Hydration. 
Nutr Rev. 2012;70 Suppl 2:S137-142). Throughout each intervention, fluid status was 
monitored via changes in body mass using a high-performance digital weighing terminal 
(model CBU150X, Mettler Toledo Inc).  
Additional whole blood (not used in PMBC extraction) will be used to measure 
haematological parameters in duplicate (Ac·T diff, Beckman Coulter). Haemoglobin and 
haematocrit will be used to estimate changes in plasma volume from the start to end of 
the 9-hour exposure (Dill & Costill. J Appl Physiol. 1974;37(2):247-248).  
Statistical analysis plan 

 
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Intervention 1 
Statistical analysis for Intervention 1 will compare whole-body heat storage and the 
resultant progression of body temperature, cardiovascular and acute inflammatory 
responses between the young and older adults during the simulated heatwave. Whole-
body heat storage (primary outcome), body temperature responses including rectal 
temperature (primary outcome) and mean skin temperature (secondary outcome), as well 
as cardiovascular responses (secondary outcomes) will be evaluated using linear mixed-
effects models. Time will be modelled as a repeated within-subject fixed effect (two levels: 
3 and 9 hours), and age-group (two levels: young and older adults) will be modelled as a 
between-subject fixed effect. Pre-heat exposure values of the outcome variable, 
participant sex, and self-reported weekly physical activity (min/week, as assessed via the 
GAQ) will be included as covariates. Circulating and intracellular markers of acute 
inflammation and stress (secondary outcomes) will be evaluated using a similar model 
but without the fixed effect of time (data was only collected at the end of exposure) and 
baseline values included as a covariate (where appropriate). Participant identification will 
be modeled as a random effect in all analyses. Akaike's information criterion will be used 
to determine random effect and variance/covariance structures.  
Post hoc multiple comparisons will be made on model estimated marginal means. Given 
the small number of comparisons for each variable, multiplicity corrections will not be 
employed. Homoscedasticity will be evaluated for all models by visual assessment of 
residual plots. Approximate normal distribution of residuals will be assessed via visual 
inspection of histograms and Q-Q plots. Data will be log-transformed in the event that the 
distribution of residuals meaningfully deviates from normality. For all analyses, the level 
of significance will be set at P < 0.050. Descriptive statistics will be presented as means 
and standard deviations. Comparisons between groups and/or time-points will be 
presented as means and 95% confidence intervals [lower limit, upper limit].    

 
16 
Intervention 2 
Statistical analysis for Intervention 2 will evaluate the acute and lasting impacts of the 
short-term cooling intervention (i.e., during cooling and following return to the heat, 
respectively) on physiological responses. 
Cumulative heat storage (primary outcome) 
The effect of the cooling intervention on cumulative whole-body heat storage over the 3 
hours following return to the heat will be evaluated using a linear mixed-effects model 
with experimental group (two levels: cooling and no-cooling) modelled as a between-
subject fixed effect. Cumulative heat storage over the first three hours of the 9-hour 
exposure will be included as a covariate to account for the influence of any inter- or intra-
individual factors (e.g., sex, physical activity levels), measured or unmeasured, impacting 
whole-body heat exchange and storage. Comparisons of heat storage between groups 
will be made using model estimated marginal means.  
Body core temperature (primary outcome) 
Body core temperature, as estimated by rectal temperature, will be analyzed with a linear 
mixed effects model with experimental group (two levels: cooling and no-cooling) 
modelled as a between-subject fixed effect and time as a repeated within-subject fixed 
effect (0-, 1-, 2-, and 3-hours post-cooling intervention). Like the model for heat storage, 
rectal temperature at the end of the first calorimetry session (i.e., hour 3 of the 9-hour 
exposure) will be included as a covariate to account for the influence of any measured or 
unmeasured inter- or intra-individual factors impacting the body temperature responses 
to resting heat exposure.   
Because this study was designed to assess the hypothesis that body core temperature 
will be similar (equivalent) between groups following return to the heat, the effect of 
cooling on rectal temperature will be assessed via two one-sided tests (see Lakens. Soc 
Psychol Personal Sci. 2017:8 (4), 355-362) performed on the mixed-effects model derived 
estimated marginal means at each timepoint. For the equivalence testing, the null 
hypothesis is taken as a meaningful difference between groups and its rejection therefore 

 
17 
corresponds to an equivalence of means. These are presented alongside the results of a 
traditional Welch’s t-test. This permits evaluation of both statistical differences, and 
statistical equivalencies in physiological responses. Performing analysis on the marginal 
means will allow for isolation of the effect of cooling from individual differences in the body 
temperature response to heat exposure (captured in the 3-hour timepoint covariate). 
Equivalence bounds will be set to ±0.3°C, which corresponds to the typical day-to-day 
variation of core temperature (Consolazio  et al. Physiological measurements of metabolic 
functions 
in 
man. 
1963:453-480) 
and 
has 
been 
suggested 
to 
reflect 
a 
meaningful/detectable change in body temperatures in a recent study assessing the 
influence of cooling strategies on physiological strain in young adults (Morris et al. Annals 
of Internal Medicine. 2019;171(9):675-677).  
Secondary outcomes 
Mean skin temperature as well as cardiovascular responses and circulating/intracellular 
inflammatory/stress markers will be modeled with linear mixed-effects models. The 
analyses for mean skin temperature and cardiovascular responses will be performed 
similar to that for rectal temperature except only two time points will be included (0- and 
3-hours post-cooling intervention). Circulating and intracellular changes in inflammatory 
markers, measured at the start and end of exposure only, will be analyzed with a linear 
mixed-effects model with age-group (two levels: young and older adults) modelled as a 
between-subject fixed effect, and participant sex, self-reported and weekly physical 
activity (min/week, as assessed via the GAQ), included as covariates. 
The analysis of secondary variables will focus on whether there was a lasting effect of 
cooling 3 hours following return to the heat. However, we will also perform tertiary 
exploratory analysis to evaluate whether the end-exposure responses are similar 
(equivalent) within pre-specified bounds. Describing clinically meaningful bounds in this 
context is complex since there is little research regarding how the risk of adverse health 
events changes with acute alterations in cardiovascular responses or inflammatory status 
during heat exposure (or cooling). For heart rate (± 5 beats/min) and rate pressure product 
(± 1500 beats/min), the equivalence bounds for these exploratory analyses will be based 
on meaningful/detectable differences employed in recent studies evaluating the efficacy 

 
18 
of personal cooling strategies in laboratory-based studies (Morris et al. Annals of Internal 
Medicine. 2019;171(9):675-677). For arterial blood pressure responses, we will consider 
± 5 mm Hg an appropriate clinically meaningful bound, given the associated 9%, 13%, 
and 6% reduction in risk of myocardial infarction, stroke, and mortality (Psaty et al. Arch 
Intern Med. 2001;161(9):1183-1192). For the other variables, reasonable equivalence 
bounds are not forthcoming. We will therefore set the bounds for these outcomes at ±1 
SD of the between-group difference (based on our sample size determination). 
For all models, participant identification will be modeled as a random effect and Akaike's 
information criterion will be used to determine random effects and variance/covariance 
structure. The level of significance will be set at P < 0.050.  Descriptive statistics will be 
presented as mean (standard deviation). Both 90% confidence intervals and 95% 
confidence intervals will be computed to facilitate interpretation of the equivalence testing 
and null hypothesis significance testing (respectively).  
As noted at the start of the document, the statistical analysis plan was submitted after 
cleaning and analysis of the full data set, but prior to unblinding of the data. 
Delimitations and limitations 
All participants recruited for this research will be relatively healthy, sedentary, or habitually 
active but non-endurance trained adults aged 18-80 years. The results may not be directly 
applicable to different population groups (e.g., children, adolescents or the extremely old 
[80+ years], the sick). Although an approximately equal number of males and females will 
be recruited for each intervention to ensure a sample representative of the general 
population, the experiments performed will not permit the assessment of the interaction 
of age and sex on thermoregulatory and cardiovascular responses to long-duration heat 
stress. Finally, behavioral factors in large part determine one’s risk of mortality and 
morbidity during heatwaves. While the project will provide novel information on the 
physiological basis of heat tolerance, defining risk is a multi-faceted endeavour and 
thereby larger in scope than any one series of studies in a given domain.