Health Canada - Establishing Evidence-based Indoor Temperature Thresholds to Protect Health
Phase: NA
Conditions: Aging, Heat Stress, Physiological Stress
Sponsor: University of Ottawa

 
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STUDY PROTOCOL AND STATISTICAL ANALYSIS PLAN 
 
 
Project: Establishing Evidence-based Indoor Temperature Thresholds to 
Protect Health (2018-026H) 
 
 
Prepared by: Robert D. Meade1, Ashley P. Akerman1, and Glen P. Kenny1,2 
 
1Human and Environmental Physiology Research Unit, School of Human 
Kinetics, University of Ottawa, Ottawa, Ontario, Canada,  
 
2Clinical Epidemiology Program, Ottawa Hospital Research Institute, 
Ottawa, Ontario, Canada.   
 
 
NCT number:  
 
Date uploaded: April 13th, 2020 
 
 
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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Relevance of the research project 
Multiple epidemiological reports have detailed the relations between outdoor 
temperature and mortality and morbidity. These generally demonstrate that older adults 
are among the most at risk of adverse health events during heatwaves. Concurrently, 
laboratory-based physiology studies have demonstrated that older adults exhibit impaired 
thermoregulatory and cardiovascular responses to heat exposure compared to their 
younger counterparts. The majority of these studies have exposed participants to extreme 
ambient heat stress. However, most people, especially older adults, spend ≥70% of their 
time in the home, where temperatures can range anywhere from ≤22°C, if the home is 
actively cooled, to ≥35°C in the case of insulated and poorly ventilated domiciles, even 
for those living in temperate continental climates.  
To protect individuals against heat stress in the home, a recent report published 
by Toronto Public Health recommended that the maximum indoor temperature limit for 
multi-unit residential buildings should be set at 26°C. The World Health Organization also 
provides recommendations for maximal indoor temperatures, though their threshold 
temperature is set considerably higher at 32°C. Importantly, much of the evidence used 
to support these guidelines is indirect, based primarily on thermal comfort and relations 
between mortality and outdoor temperatures. Moreover, both guidelines offer a ‘one-size-
fits-all’ solution, such that neither explicitly considers the thermoregulatory and 
cardiovascular insufficiencies that may reduce the range of tolerable indoor conditions for 
vulnerable populations (e.g., older adults). Despite these limitations, the thermal and 
cardiovascular strain experienced in conditions representative of indoor environments, 
where older adults spend the majority of their time, has not been directly evaluated. 
 
Research Project Objectives 
The objective of this project is to evaluate the effect of a range of environments 
experienced during heatwaves on body temperature and autonomic cardiovascular 
function in older adults. This will be accomplished by comparing thermal and 
cardiovascular responses in a group of older adults resting for 8-hours in a range of 
conditions representative of those experienced indoors during heatwaves – from 
insulated and poorly ventilated domiciles (36°C), through currently recommended indoor 

 
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temperature thresholds set by the World Health Organization (32°C) and Toronto Public 
Health (26°C), to an actively-cooled environment (22°C). To assess how the strain 
experienced in these conditions affects haemodynamic regulation in turn, this study will 
also include clinically validated tests of cardiovascular function selected to mimic 
autonomic challenges experienced during activities of daily living (e.g., quickly standing 
from a lying position). Combining resting measurements of physiological strain with 
validated autonomic functional tests will allow for the assessment of i) the level of 
hyperthermia experienced by older adults resting in relatively cooler ambient conditions 
(e.g., indoor environments), and ii) how the associated hyperthermia may in turn impact 
the regulation of cardiovascular function and haemodynamic stability. The latter objective 
is of particular importance given that most acute injury during heatwaves are 
cardiovascular in origin.  
 
Hypotheses 
Indices of thermal and cardiovascular strain will not be appreciably modified in older 
adults resting in simulated indoor environments below the currently recommended 
thresholds set by Toronto Public Health (22°C and 26°C). However, at higher indoor 
temperatures (31°C and 36°C) thermal (rectal temperature) and cardiovascular strain 
(heart rate, rate pressure product) will be elevated compared to below-threshold 
conditions. The increased physiological strain in these conditions will be reflected in 
attenuated responses to validated cardiovascular autonomic functional tests.   

 
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Methods 
Participants 
A total of 16 older (age: 65-80 years) adults will be recruited for the proposed 
project with an approximately even distribution of men and women. Each participant will 
complete each of the 4 experimental arms of the study. Recruited participants will be 
homogenous for anthropomorphic characteristics as well as habitual physical activity 
levels as verified via standardized questionnaires. All participants will be non-smoking 
and will be screened for electrophysiological abnormalities. Written and informed consent 
will be obtained from all volunteers prior to participation.  
 
Experimental Design 
Pre-trial instructions  
All participants will be asked to avoid strenuous 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. 
For the experimental sessions, hydration state will be verified upon arrival to the 
laboratory via urine specific gravity (euhydration operationally defined as a urine specific 
gravity <1.025). 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. Participants 
will wear an athletic shirt and shorts. For the preliminary session, participants also will 
wear an athletic t-shirt.  
 
Preliminary screening 
 
All participants will complete one preliminary evaluation a minimum of 48 hours 
before the first experimental session. During this session they will be familiarized with all 
experimental procedures and measurement techniques and will complete the 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. Participants 
will also provide verbal and written informed consent at this time, Thereafter, participant 

 
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physical characteristics will be evaluated. Body height and mass will be determined via a 
physician stadiometer and a high-performance weighing terminal, respectively, and from 
these measurements body surface area will be calculated. Body density will be estimated 
via hydrostatic weighing and used to calculate body fat percentage. 
 
Experimental Protocol 
Experimental design 
Each session will commence at approximately 08:00. Upon arrival to the laboratory 
the participant will provide a urine sample for measurement of urine specific gravity and 
insert a rectal temperature probe. Thereafter, participants will be instrumented for the 
measurement of skin temperature. A measurement of body mass (nude) will also be taken 
at this time. Participants will then be transferred to a thermal chamber regulated to 22°C 
and 45%. Following 10-min of supine rest, participants will perform a specialized 
cardiovascular test battery (~75 min; details provided below). Following this, they will rest 
in the seated position in a room immediately adjacent to the thermal (~22°C) chamber for 
~30 min while baseline body temperature (rectal temperature, skin temperature) and 
cardiovascular measures (heart rate, arterial blood pressures) are recorded. A venous 
blood sample will be obtained at this time.   
While baseline measurements are being taken, the thermal chamber will be heated 
to the conditions for that day’s session. Each participant will complete four separate 
(separated by ≥7 days) sessions consisting of resting exposure to: i) 36°C, based on 
maximal measured indoor temperatures (i.e., 35°C) and similar to outdoor conditions 
experienced during recent heatwaves in North America (HOT, heat index: 41°C); ii) 31°C, 
similar average daytime temperature during the 2010 heatwave in Ontario and Quebec 
as well as the World Health Organization recommended indoor temperature limit during 
heatwaves  (WARM, heat index: 32°C); iii) 26°C corresponding to recommended upper 
limits for indoor environments as set by Toronto Public Health based before protective 
cooling measures should be implicated (TEMPERATE, heat index: 26°C) and iv) 22°C, 
to simulate an air-conditioned environment (COOL, heat index: 21°C). In all sessions, 
relative humidity will be regulated to 45%. 

 
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Following baseline measurements, the participant will enter the thermal chamber 
where they will remain for 8 hours. They will spend the majority of this time resting in the 
seated (slightly reclined) position, except for at the 6th hour where they will again perform 
the cardiovascular test battery. Rectal and skin temperatures and heart rate will be 
measured continuously throughout exposure. Water (tap) will be provided ad libitum from 
a ~500 mL cup placed on a side-table positioned slightly behind the participant (so as not 
to be visible in the peripheral vision). Participants will also be allowed to eat a self-
provided lunch (~300 g, low water content) between hours 1.5 and 3.5 of exposure 
(corresponding to ~12:00-14:00 hh:mm). Every 60 min, arterial blood pressure and heart 
rate will be measured in triplicate after which participants will stand to provide a 
measurement of body mass in order to determine sweat losses (corrected for consumed 
food and water). Participants will be allowed to remain standing and briefly perform light 
stretching, if desired (max ~5 min).  
Autonomic control of heart rate and blood pressure will be assessed prior to and 
at the end of each session using a battery of cardiovascular tests, which, in Study B 
consists of: i) analysis of heart rate variability and spontaneous baroreflex sensitivity 
during supine rest, ii) Ewing’s battery (modified) for assessment of cardiovascular 
autonomic function, and iii) analysis of integrated baroreflex sensitivity during cyclical 
perturbations in arterial pressure evoked by stand-squat maneuvers. Prior to the battery, 
participants will be instrumented with an integrated 3-lead ECG non-invasive blood 
pressure monitor for the integrated measurement of beat-to-beat heart rate and arterial 
blood pressure, estimated from the arterial pressure waveform measured at the left 
middle finger using the volume-clamp technique. Throughout the battery, the arm will be 
affixed to the chest using a medical sling with the finger held at the approximate level of 
the left ventricle. Participants will be instructed to breathe normally and, without speaking, 
carefully follow the instructions given by the test administrator. The same researcher will 
administer the battery for all sessions. Finally, note that the first two sections of the battery 
described below (i.e., resting baroreflex sensitivity and Ewing’s test) will be performed 
twice in succession prior to the stand-squat procedures.  
i. 
Resting HRV and BRS. Heart rate variability (HRV) and baroreflex sensitivity (BRS) 
will be evaluated during 8-min of supine rest. Participants will be instructed to breathe 

 
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(diaphragmatic) in time with a metronome set to 30 beats/min (15 breaths/min), to 
remove the influence of respiratory sinus rhythm on cyclical fluctuations in heart rate 
and blood pressure. Thereafter, the metronome will be stopped and 1-min of recovery 
(spontaneous breathing) will be given prior to the start of the Ewing’s battery.   
ii. 
Ewing’s battery. The modified Ewing’s battery to be employed consists of three tests 
(deep breathing, Valsalva’s maneuver and lying-to-standing test) designed to assess 
autonomic control of heart rate (primarily parasympathetic) and blood pressure 
(primarily sympathetic).  
• First, participants perform 1 min of deep diaphragmatic breathing at a rate of 6 
breaths·min-1 (metronome set to 12 beats/min). Breathing will be performed to vital 
capacity; that is, through a maximal inspiration and expiration. 5-min of recovery will 
be given before the next test. 
• The next test is the Valsalva maneuver. While still in the supine position, participants 
will exhale against a closed glottis into a narrow vinyl tube connected to an electronic 
pressure transducer that will be used to provide constant feedback to the participant 
on the generated expiratory pressure. The participant will be instructed to maintain 
a constant pressure of 40 mmHg for 15 s. Throughout expiratory straining, air will 
be vented via a small bleed to prevent generation pressure using the mouth. Upon 
release, heart rate and blood pressure will be monitored for 1 min.  
• The final test in Ewing’s battery is the lying-to-standing test. The test begins with 1-
min of rest in the supine position. At this point, the participant is instructed to quickly 
(but safely) assume a standing position directly beside the bed (max ~1 min). The 
participant then stands quietly for 3 min.  
iii. 
Squat-stand. Following the second Ewing’s battery, the participant will rest in the 
seated position on a rigid chair for 10 mins. The participant will then be asked to stand 
for 90-seconds while the positioning of the sling and finger-pressure cuff is adjusted (if 
needed) to ensure the left hand is maintained at heart-level. After this, 5-min of squat-
stands will be performed at a rate of 6 cycles/min (0.1 Hz).  Each cycle will consist of 
a squat, held for 5 seconds, followed by standing for 5 sec. For these maneuvers, 
participants will be instructed to maintain a consistent depth for each squat (based on 
comfort), breath normally to avoid expiratory straining (Valsalva) and limit excessive 

 
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forward flexion at the hip. Participants will be verbally aided in timing for the first ~2-3 
cycles and self-timed thereafter by a stopwatch located directly within their line of sight.  
After the cardiovascular battery, participants will remain seated in the chamber for 
the remainder of the 8-hour exposure (~45 min). Following measurements of arterial 
blood pressure, a final venous blood sample will be obtained followed by a measurement 
of body mass. The participant will be provided with water and/or a commercially available 
sports drink before leaving the laboratory.  
 
Data analysis 
Continuous variables related to thermal strain (rectal and mean skin temperatures) 
and cardiovascular strain (heart rate, rate pressure product) will be converted to 15-min 
averages for baseline and at each hour of exposure. Likewise, seated arterial pressures 
(3 measurement average) will be presented at these time points. Rate pressure product 
(an index of myocardial work) will be calculated as heart rate x systolic blood pressure x 
10-3. The change in body mass from baseline values will also be determined at each hour 
of exposure to quantify the rate of fluid loss, corrected for fluid consumed and urination. 
The change in plasma volume and serum osmolality from baseline to end exposure will 
also be calculated from the venous blood samples obtained at the start and end of each 
session. HRV and resting spontaneous cardiac BRS will be determined from data 
collected during the 8-min controlled breathing period. Similarly, integrated BRS will be 
derived from collected blood pressure and ECG waveforms collected during the stand-
squat manoeuvres.   
For the clinically validated tests in the Ewing battery, analysis will be performed by 
a trained researcher blinded to the participant and session code (i.e., environmental 
conditions). Deep breathing will be analysed as the difference in heart rate between the 
average of the three lowest and three highest values recorded during inspiration and 
expiration, respectively (E/I HRDiff). For the Valsalva maneuverer, the so-called Valsalva 
ratio (VALratio) is calculated as the longest R-R interval during recovery divided by the 
shortest R-R interval during straining. Finally, the lying to standing test is analysed by 
determination of the 30:15 ratio, which represents the longest RR interval measured 
between the 25-35th heart beat upon standing divided by the shortest RR interval between 

 
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the 10-20th beat after standing. Additionally, the systolic response to standing (SRstand) is 
also taken as the 1-min average of systolic blood pressure after the second min of 
standing divided by systolic blood pressure during the pre-stand supine resting period 
(i.e., the reduction in systolic pressure upon assuming a standing position). The 
participant identifier and experimental arm will be blinded from the investigator 
responsible for analyzing outcomes in the cardiovascular battery. Values for each test will 
be taken as the average of each of the two recorded values. 
 
Statistical analysis and sample size calculation 
Statistical analysis will compare end-exposure data relating to thermal and 
cardiovascular strain as well as autonomic function (while correcting for between-session 
baseline variability). Body temperature (i.e., rectal temperature and mean skin 
temperature) and cardiovascular responses (i.e., systolic, diastolic and mean arterial 
pressure, heart rate, rate pressure product) at the end of exposure (i.e., hour 8) as well 
as the autonomic functional tests of the cardiovascular battery (i.e., E/I HRDiff, VAL ratio, 
30:15 ratio and SRstand) will be evaluated using an analysis of covariance (ANCOVA) with 
the repeated factor of environmental condition (four levels: HOT, WARM, TEMPERATE 
and COOL) and baseline values modelled as a continuous covariate. Variables related to 
fluid loss (e.g., change in body mass, plasma volume and serum osmolality) will be 
analysed via a one-way ANOVA with the factor of condition (4 levels). For all statistical 
models, homogeneity of variances will be assessed using Levene’s test and visual 
assessment of residual plots. Normal distribution of residuals will be assessed with 
Sharpiro-Wilks test and visual inspection of histograms and Q-Q plots and data will be 
log-transformed in the event that distribution of residuals deviates from normality. Pre-
planned post hoc comparisons will be employed to identify differences between adjacent 
conditions (e.g., COOL vs TEMP, TEMP vs. WARM, WARM vs. HOT) in order to limit a 
large family of comparisons. For all analyses, alpha will be set at P < 0.050. Descriptive 
statistics will be presented as mean (standard deviation). Comparisons between groups 
or time-points (where appropriate) will be presented as means ± 95% confidence interval.   
An a priori power analysis determined that a total sample size of 16 older adults is 
required to detect a difference in rectal temperature between groups with 80% statistical 

 
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power, after adjusting for multiple comparisons. The effect size (Cohen’s d=1.0) was 
determined based on practically meaningful difference of 0.3°C between conditions, 
assuming a standard deviation of 0.3. The standard deviation was based on previously 
published studies from our laboratory, which demonstrated a 0.2°C (SD 0.3) difference in 
core temperature between young and older adults (Kenny GP et al. Temperature (Austin). 
2016 31;4(1):79-88) and a 0.0°C (SD 0.3) difference in core temperature between older 
adults with and without type 2 diabetes (Poirier et al. Temperature (Austin). 2020 [in 
press]) following 3 hours of rest in a hot environment (40°C, 30% relative humidity). 
 
Measurements 
Primary Outcome measure 
Body temperatures 
Rectal temperature will be continuously monitored using a general-purpose 
thermocouple temperature probe (Mon-a-therm General Purpose Temperature Probe, 
Mallinckrodt Medical Inc., St-Louis, MO, USA) inserted ~12 cm past the anal sphincter. 
Skin temperature will also be assessed using surface temperature monitors (DS1922L 
Thermochron, OnSolution Pty Ltd, Australia) placed in 8 locations as described in ISO 
9886:2004. Mean skin temperature will be subsequently 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%).  
 
Secondary Measures 
Cardiovascular measurements  
Heart rate will be recorded continuously throughout each experimental session and 
stored every second using a Polar coded WearLink and transmitter, Polar RS400 
interface and Polar Trainer 5 software (Polar Electro, Kempele, Finland). Arterial systolic 
and diastolic blood pressures will be intermittently taken as an average of 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 evaluated as heart rate 
× systolic pressure x 10-3. Brachial systolic and diastolic arterial blood pressures will also 

 
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be estimated from beat-to-beat recordings of arterial pressure waveform measured at the 
left middle-finger using the volume-clamp technique (NIBP, ADInstruments, Colorado 
Springs, CO, USA). In the specialized cardiovascular test battery, beat-to-beat arterial 
blood pressure measures will be coupled with beat-to-beat heart rates derived from R-R 
interval measured via an integrated 3-lead ECG (FE231 Bio Amp, ADInstruments, 
Colorado Springs, CO, USA).  
 
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, Depew, 
NY, USA). Participants presenting to the laboratory with urine specific gravity <1.025 will 
be considered adequately hydrated to begin the experimental session. Throughout each 
intervention, fluid status will be monitored via changes in body mass. Pre and post heat 
exposure measurements will be obtained using a high-performance digital weighing 
terminal (model CBU150X, Mettler Toledo Inc., Mississauga, ON, Canada). This unit will 
also be used to monitor changes in body mass throughout the intervention.  
Venous blood samples will be collected for determination of plasma volume and 
serum osmolality responses. Blood samples will be transferred directly into plasma (5.4 
mg K2EDTA) and serum (no additive) Vacutainer tubes (BD, Franklin Lakes, NJ, USA). 
The K2EDTA blood is mixed by inversion and used to measure haematological 
parameters in duplicate (Ac·T diff, Beckman Coulter, Miami, FL, USA). Measured blood 
haemoglobin and haematocrit will be used to estimate changes in plasma volume. Non-
additive blood is left to sit for 20 min to clot before centrifugation (~1380 g for 10 min). 
Separated serum is then transferred into polypropylene Eppendorf tubes, frozen at -20ºC, 
and stored at -80ºC until serum osmolality can be later analyzed via freeze-point 
depression (model 3320, Advanced Instruments, Norwood, MA, USA).  
 
Delimitations and limitations 
All participants recruited for this research will be sedentary or habitually active but 
non-endurance trained adults aged 65-80 years. The results may not be directly 
applicable to different population groups (e.g., children, adolescents or the extremely old 

 
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[80+ years]). Although an approximately equal number of men and women 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, it is important to consider that defining risk is a multi-
faceted endeavour and thereby larger in scope than any one series of studies in a given 
domain.