Characterizing the Repeatability and Reproducibility of Cardiovascular Responses to Hypoxic Apneas
Phase: NA
Conditions: Hypoxia, Apnea
Sponsor: University of Alberta

 
 
Characterizing the Repeatability and Reproducibility of Cardiovascular Responses to Hypoxic 
Apneas 
 
NCT06399575 
 
June 26, 2024 
 
 

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Characterizing the Repeatability and Reproducibility of Cardiovascular Responses to Hypoxic 
Apneas 
Authors: 
Desmond Young, BScKIN1 
Craig D Steinback, PhD1 
 
Programs: 
 
 
 
1Faculty of Kinesiology, Sport, and Recreation 
 
PI: 
Craig D Steinback, PhD 
 
 
Institutions: 
University of Alberta 
 
Faculty of Kinesiology, Sport, and Recreation 
1-052 Li Ka Shing Centre for Health Research Innovation 
8602-112 St  
TELE: 780-492-5553; FAX: 780-492-4249 
craig.steinback@ualberta.ca  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CONFIDENTIAL 
This document is confidential and the property of individual.  No part of it may be transmitted, 
reproduced, published, or used by other persons without prior written authorization from the author. 
 
Protocol/version #: Pro00138947 
Current Version Date:  June 26, 2024 
 
 
 

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Table of Contents 
1. Introduction/Significance ...................................................................................................................... 3 
2 Study Objectives .................................................................................................................................... 3 
2.1 Objectives ........................................................................................................................................ 3 
2.2. Hypotheses ..................................................................................................................................... 3 
3 Participants and Methods ....................................................................................................................... 4 
3.1 Study Design ................................................................................................................................... 4 
3.2 Subject Selection ............................................................................................................................. 4 
3.3 Study Procedures ............................................................................................................................. 4 
3.3.1 Pre-testing Instructions ............................................................................................................. 4 
3.3.2 In-lab Testing ............................................................................................................................ 5 
3.3.3 Measurements ........................................................................................................................... 8 
4 Statistical Analysis Plan ......................................................................................................................... 9 
4.1 Data Analysis .................................................................................................................................. 9 
4.2 Statistical Methods ........................................................................................................................ 10 
4.3 Sample Size Determination ........................................................................................................... 11 
5 References ............................................................................................................................................ 12 
 
 
 

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1. Introduction/Significance 
 
Reproducibility of findings is a critical scientific principle required to make clear interpretations from 
results. To date, no study has tested the reproducibility of cardiovascular (i.e., heart rate and blood 
pressure) responses to apneas. Notably, previous research studies employing apneas (e.g., Busch et al., 
2018; 2020; 2021) base their conclusions on results from one apnea per person, or one apnea per 
condition per person when multiple conditions are included. These methodological decisions mean it 
remains unclear whether a second apnea would cause the same effect, necessitating an investigation to 
quantify the consistency of an individual’s response to apneas. Apneas are commonly utilized in pre-
clinical human research as an autonomic nervous system stressor. The frequency with which this 
stimulus is utilized in research justifies the need to determine the repeatability and reproducibility of 
apnea responses. 
 
2 Study Objectives 
 
2.1 Objectives 
 
The primary objective of this study is to determine the degree of repeatability (i.e., consistency within a 
testing session) and reproducibility (i.e., consistency between testing sessions) of heart rate and blood 
pressure responses to apneas. This study also aims to determine the nature of inconsistency in these 
cardiovascular responses to apneas; we will ascertain whether inconsistency stems from random 
variability or systematic changes with repeated apneas. 
 
2.2. Hypotheses 
 
We hypothesize that heart rate responses to apneas are repeatable (within-subject standard deviation 
[WSSD] <5 bpm; see Section 4.2 below for further details) and that greater temporal space between 
apneas will increase the WSSD. We also hypothesize that blood pressure responses to apneas are 
repeatable (WSSD <4 mmHg). Additionally, we hypothesize that inconsistency between apneas will 
primarily stem from random variability. 
 

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3 Participants and Methods 
 
3.1 Study Design 
 
General Design:  
We will evaluate the cardiovascular response to two maximal apneas and five repeated test apneas 
under standardized hypoxic conditions. Participants will complete two identical testing sessions 24 
hours apart. 
 
Recruitment:  
Participant recruitment will follow non-probability quota sampling and will be facilitated by word of 
mouth by the study coordinator, as well as through posters or social media posts on designated 
accounts. All potential participants will be given a copy of the informed consent document to read 
through before agreeing to participate.  
 
3.2 Subject Selection 
 
We will recruit 20 healthy volunteers (male and female) between the ages of 18 and 70.  
 
Inclusion Criteria:   
• Males and females between the ages of 18-70.  
 
Exclusion Criteria:   
• Any individual having any known cardiovascular or nervous system disease. 
• Anyone with low or high blood pressure (< 90/60 or >139/85, respectively). 
• Anyone taking any prescribed medications (other than oral contraceptives) than may affect 
cardiovascular system function.   
• We will exclude females who may be pregnant, as self reported. 
• Anyone currently involved in any other studies. 
 
3.3 Study Procedures 
3.3.1 Pre-testing Instructions   
Participants will undergo two in-lab testing sessions beginning at the same time of day on consecutive 
days. Participants will arrive at the laboratory after fasting for two hours; they will also abstain from 
caffeine, alcohol, and strenuous physical activity 12 hours before each visit. 
 

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3.3.2 In-lab Testing 
Questionnaire:  
Participants will review the consent form with a researcher and sign the form before participating in 
any testing. Participants will then be asked to fill out a Health History Questionnaire that asks about 
their medical history. 
 
Anthropometrics:  
We will measure the participant’s height and weight. 
 
Baseline Assessments:  
Participants will perform a vital capacity maneuver to provide a measure of the maximum amount of 
air they can exhale; we will use this as a measure of functional lung volume. Participants will complete 
three vital capacity tests, and the highest of two values within 150mL will be that participant’s vital 
capacity. We will then calculate 40% of this value as the amount of air participants will inhale, after a 
full breath out, immediately before each apnea (see Apnea Procedure below for further details).  
Physiological Testing:  
Figure 1 shows a schematic of the in-lab protocol. Each test will begin with a 10-minute baseline 
period where participants breathe through a mouthpiece to determine resting values for heart rate, mean 
arterial pressure, and end-tidal partial pressures of oxygen and carbon dioxide. Participants will then 
complete a single warm-up apnea to familiarize themselves with the sensations of an apnea. Next, 
participants will breathe a hypoxic gas mixture for five minutes before completing a maximal apnea. A 
dynamic end-tidal forcing system will decrease end-tidal oxygen to 50 mmHg, corresponding to 80-
85% peripheral oxygen saturation, and will maintain end-tidal carbon dioxide at baseline levels. 
Participants will complete two such apneas with a five-minute rest between the end of the first apnea 
and the beginning of the second hypoxic period. The longest of the two apnea durations will be used as 
the target time for all subsequent test apneas, rounded to the nearest second. Participants will be 
blinded to their apnea duration for the first maximal apnea to encourage a maximal effort without 
contributions from preconceived ideas about their apnea abilities. Participants will then be told their 
first maximal apnea time and be provided an opportunity to improve their maximal time with a live 
view of a stopwatch during their second apnea. Immediately following each apnea, participants will 
rate the difficulty of the apnea using a 10-point modified Borg Scale adapted from Foster et al. (2001) 
(Figure 2). 
Finally, participants will perform five successive test apneas, each after a five-minute hypoxic 
stimulus. Apnea duration for all five test apneas will correspond to the same duration as the longest of 
the two maximal apneas. Participants will see a live stopwatch so they are aware of the remaining 
duration of their apneas. They will be instructed prior to the test apneas to hold their breath for the pre-
specified duration to their best ability. Participants will get at least five minutes’ rest between the end 
of the prior apnea and the beginning of the subsequent hypoxic period. Participants will only proceed 
to a hypoxic period once heart rate and mean arterial pressure return to within five units of their 
baseline values. 

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On the second testing day, the maximal apneas will be used only for a measure of improvement from 
the first day. The test apneas on the second testing day will use the same standardized time as 
determined on the first testing day. On the second day we will also set the end-tidal partial pressure of 
carbon dioxide to the baseline value from the first day; this will eliminate any differential effects of 
carbon dioxide on day-to-day reproducibility. 
 
Figure 1. Protocol schematic. Participants will begin with a vital capacity test and instrumentation. 
They will then complete a warm-up apnea. The hourglass and symbol above refer to whether the 
participant will see their apnea duration in live time. Next, participants will complete two maximal 
apneas; the longest apnea duration will be the time used for the five test apneas. Participants will 
complete the same in-lab protocol on consecutive days. On the second day the protocol is the same 
except participants do not complete another vital capacity test, and the test apnea time is the same as 
the first day, regardless of the maximal apnea times. SpO2 peripheral oxygen saturation, ECG 
electrocardiogram, BP blood pressure, AirForce refers to the dynamic end-tidal forcing system. 
 
 

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Rating 
Descriptor 
0 
Rest 
1 
Very, Very Easy 
2 
Easy 
3 
Moderate 
4 
Somewhat Hard 
5 
Hard 
6 
. 
7 
Very Hard 
8 
. 
9 
. 
10 
Maximal 
Figure 2. Modified Borg scale for rating of perceived exertion. Reproduced from Foster et al. (2001). 
Apnea Procedure 
Figure 3 shows a schematic of the mouthpiece setup. We will arrange the mouthpiece setup through 
which the participant will breathe as follows: the mouthpiece will connect to a filter, which will further 
attach to a pneumotachometer and a three-way non-rebreathing valve, all in series. The expired port of 
the non-rebreathing valve will be open to the room, and the inspired port will connect to a second 
three-way valve. Port 1 of the valve will attach to the mouthpiece setup, port 2 will attach to the 
dynamic end-tidal forcing system, and port 3 will attach to a standard three-liter calibration syringe 
(Model 5530, Hans Rudolph). The three-way valve has three positions during different phases of the 
protocol. Position 1 allows flow between ports 1 and 2; position 2 allows flow between all three ports; 
and position 3 allows flow between ports 1 and 3. Three researchers will work together during the 
apnea procedure: the first will control the dynamic end-tidal forcing system, the second will switch the 
positions of the three-way valve (valve operator), and the third will manage the volumes of a three-liter 
calibration syringe (syringe operator). The procedure begins with the valve in position 1 for the 
duration of the hypoxic step. Thirty seconds before the apnea, the syringe operator attaches an empty 
standard three-liter calibration syringe to port 3, and the valve operator subsequently switches the valve 
to position 2, allowing flow between all three ports. Next, the operator of the dynamic end-tidal forcing 
system adds gas corresponding to 40% of the participants’ vital capacity (in addition to tidal volume) 
over one or two breaths, depending on tidal volume, and the syringe operator pulls gas into the syringe 
to the correct volume, rounded to the nearest 100 mL. Finally, the valve operator will instruct the 
participant to exhale fully; during the exhalation the valve operator will switch the valve to position 3 
to isolate the syringe and the participant. The participant will then inhale from the syringe until an 
audible clunk, at which time the valve operator will assist in removing the mouthpiece, and the 
participant will hold their breath with their mouth closed. Since the dynamic end-tidal forcing system 
alters inspired gas concentrations on a breath-by-breath basis, the syringe system standardizes apnea 
lung volume while continuing to target the desired end-tidal partial pressures of oxygen and carbon 
dioxide on the final breath in a participant-specific manner. 
 

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Figure 3. Mouthpiece setup during the hypoxic periods. The orange and black numbers on the three-
way valve correspond to the port numbers, and the port to which the orange and black arrow points 
corresponds to the position of the valve (see Apnea Procedure for additional details). 
3.3.3 Measurements  
Respiratory Measures:  
Figure 3 shows a schematic of the mouthpiece setup. Participants will wear a nose clip and breathe 
through a mouthpiece attached to a pneumotachometer (MLT3813H-V, ADInstruments) and a 3-way 
non-rebreathing valve (T-Shape Series, 2700, Hans Rudolph). The inspired port of the valve will 
connect to a dynamic end-tidal forcing system that will control end-tidal oxygen and carbon dioxide on 
a breath-by-breath basis using offline software (Labview 16.0, National Instruments). The mouthpiece 
will have a small sampling port connected to oxygen (S-3A/I Oxygen Analyzer & Model N-22M 
Sensor, AEI Technologies) and carbon dioxide (CD-3A Carbon Dioxide Analyzer & P-61B Sensor, 
AEI Technologies) gas analyzers run in parallel. The gas analyzer user manual claims accuracy within 
±0.01% O2 and ±0.02% CO2 (“Respiratory Gas Analyzers,” n.d.). The gas analyzer delays will be 
measured daily in accordance with the calibration procedures for the dynamic end-tidal forcing system. 
A respiratory strain gauge (Model TN1132/ST, ADInstruments) placed around the chest will measure 
respiratory movements during normal breathing and breath-holding and will assist in detecting the end 
of each apnea. 
 
 
 

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Cardiovascular Measures:  
We will measure heart rate and rhythm using an electrocardiogram (lead II). We will measure blood 
pressure using finger photoplethysmography (Finometer Pro, Finapres Medical Systems) calibrated to 
manual blood pressure measurements taken in triplicate during the baseline. We will measure 
peripheral hemoglobin oxygen saturation using pulse oximetry (Nellcor N-600x, Medtronics).  
 
Borg Scale: 
We will ask participants to rate the effort of each test apnea on a scale of 1-10. We will also have a 
visual modified Borg scale diagram to assist participants in rating the effort of each apnea (Figure 2). 
4 Statistical Analysis Plan 
4.1 Data Analysis 
We will collect all data at 1 kHz using LabChart software (Chart Pro, v8.1.3, ADInstruments). We will 
calculate mean arterial pressure as the arithmetic mean of the blood pressure waveform. We will also 
calculate minute ventilation as the product of breathing frequency and tidal volume; the latter is 
determined as the integral of the inspired portion of the respiratory pneumotach trace. We will then 
extract data into Microsoft Excel for offline analysis (Microsoft Inc., Washington, USA). We will 
extract free-breathing values as one-minute averages, and we will extract apnea values beat-by-beat. 
 
We will determine four different apnea heart rates and blood pressures using four distinct methods. 
First, we will use the single slowest heartbeat (i.e., nadir beat [NADIR]) within the final 10 cardiac 
cycles of an apnea. Second, we will average the heart rates between the nadir beat and the preceding 
beat (AVG2). Third, we will average the nadir beat and the four beats surrounding it (AVG5). Fourth, 
we will average the final five cardiac cycles of each apnea (AVG_END). The different apnea values 
stem from an attempt to increase repeatability and reproducibility through analyzing more data rather 
than the standard technique of one cardiac cycle per apnea. Table 1 provides a visual representation of 
the methods to determine apnea heart rate and blood pressure.  
 
We will calculate the change in heart rate as apnea heart rate minus free-breathing heart rate (a negative 
value would indicate a decrease in heart rate). We will analyze mean arterial pressure in the same 
manner as heart rate. We will calculate the minimum peripheral oxygen saturation as the lowest value 
during the apnea or within one minute following the termination of the apnea to account for the 
circulatory delay in detecting peripheral oxygenation; We will calculate the change in peripheral 
oxygen saturation as minimum minus free breathing. We will determine apnea duration using the 
airflow readings from the pneumotach at the start of apnea and the chest movement measured by the 
respiratory belt at the end of apnea. To identify arrhythmias, a trained individual (DY) will visually 
inspect the electrocardiograph during all apneas. All abnormal rhythms will be reviewed by a 
cardiologist who will verify the presence and type of arrhythmia.  
 
 

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Table 1. A visual representation of the four methods of determining apnea heart rate (HR) in beats 
per minute and mean arterial pressure (MAP) in mmHg. The single nadir beat (NADIR for HR) or 
peak beat (PEAK for MAP) is enlarged and bolded. The average of two beats (AVG2) includes the 
NADIR/PEAK beat and the preceding beat, underlined. The average of five beats (AVG5) includes 
the NADIR/PEAK beat and the four closest beats, shown in red text. The average of the five final 
beats (AVG_END) is shown highlighted in yellow. 
Cardiac Cycle 
-10 
-9 
-8 
-7 
-6 
-5 
-4 
-3 
-2 
-1 
0 
HR 
71 
70 
66 
64 
59 
54 
51 
53 
52 
55 
54 
MAP 86 
85 
88 
87 
86 
90 
89* 
92* 
96 
94 
97 
HR: NADIR=51, AVG2=52.5, AVG5=53.8 AVG_END=53 
MAP: PEAK=97, AVG2=95.5, AVG5=93.6, AVG_END=93.6 
*Note: in any instance where the NADIR/PEAK beat occurs at beat 0, -1, -9 or -10 the AVG2 and/or 
AVG5 calculations are shifted such that only beats -10 to 0 are included in any calculations. If two 
beats in the final 10 cardiac cycles of an apnea are tied for NADIR/PEAK, the beat closest to the end 
of the apnea will be used. 
 
4.2 Statistical Methods 
We will report all continuous data as mean ± standard deviation; modified Borg ratings will be reported 
as median (interquartile range). We will complete all statistical analyses in R Statistical Software 
(v4.3.1 R Core Team 2023). We will use summary statistics alongside boxplots and histograms to 
confirm the normality of continuous data. To assess absolute repeatability, we will compute within-
subject standard deviations (WSSD) for heart rate, mean arterial pressure, peripheral oxygen saturation, 
and minute ventilation. The WSSD is the average amount we can expect an individual’s future score to 
deviate from their current score. It is computed using a two-way ANOVA with the participant and 
apnea number (i.e., the identity of the apnea in a sequence of repeated measurements) as factors. The 
WSSD is the square root of the mean square of residuals (Hopkins, 2000; Weir, 2005). We will also 
calculate two-way mixed effects, absolute agreement, single measurement intraclass correlations (ICC) 
for heart rate, mean arterial pressure, peripheral oxygen saturation, and minute ventilation (Koo & Li, 
2016). We will present all arrhythmia data as summary statistics and average percent incidence within 
individuals. We will present modified Borg data as summary statistics. Because none of our analyses 
will utilize p values or rely on the outcome of a null hypothesis test per se, we do not have an a priori 
alpha for statistical significance. 
 

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4.3 Sample Size Determination 
We will recruit 20 participants to ensure we obtain 16 participants who reach their target apnea time on 
at least 4 of the 5 test apneas on both test days. McAlinden et al. (2015) presented a method for 
calculating sample size for a repeatability and reproducibility study: 
1.96
𝑊𝑆𝑆𝐷
√2𝑛(𝑚−1)
= 𝑊𝑆𝑆𝐷× 𝐿𝐶 
where n is the sample size, m is the number of repeated measurements, and LC is the desired level of 
uncertainty between the sample WSSD and the population parameter. Note that WSSD appears on both 
sides of the equation and therefore divides to one; in other words, the magnitude of the WSSD does not 
affect the sample size calculation. Rearranging the equation to isolate n, using 20% uncertainty 
between the sample estimate and the population parameter (LC = 0.20), and four repeated trials (m = 
4), we require 16 participants. Therefore, to account for instrumental challenges and incomplete trials 
we will recruit up to 20 participants to obtain a complete sample of eight males and eight females who 
will complete five apneas per trial. If all apneas go well then we will analyze the first four and discard 
the final apnea; we decided a priori that we will only use the final apnea for analysis if an apnea 
duration deviates more than two seconds from the target time, or if the heart rhythm is uninterpretable 
during analysis from excessive participant movement or any other cause. We will record and report 
every instance that we use the final apnea for analysis, and the reason for its use.  
 
 
 

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5 References 
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Ainslie, P. N., Willie, C. K., Hoiland, R., Moore, J. P., & Steinback, C. D. (2018). Chemoreflex 
mediated arrhythmia during apnea at 5,050 m in low- but not high-altitude natives. Journal of 
Applied Physiology, 124(4), 930–937. https://doi.org/10.1152/japplphysiol.00774.2017 
Busch, S. A., van Diepen, S., Roberts, R., Steele, A. R., Berthelsen, L. F., Smorschok, M. P., Bourgoin, 
C., & Steinback, C. D. (2021). Short-term hypoxia does not promote arrhythmia during 
voluntary apnea. Physiological Reports, 9(1), e14703. https://doi.org/10.14814/phy2.14703 
Busch, S. A., van Diepen, S., Steele, A. R., Meah, V. L., Simpson, L. L., Figueroa-Mujíca, R. J., 
Vizcardo-Galindo, G., Villafuerte, F. C., Tymko, M. M., Ainslie, P. N., Moore, J. P., 
Stembridge, M., & Steinback, C. D. (2020). Global REACH: Assessment of Brady-
Arrhythmias in Andeans and Lowlanders During Apnea at 4330 m. Frontiers in Physiology, 10. 
https://www.frontiersin.org/articles/10.3389/fphys.2019.01603 
Foster, C., Florhaug, J. A., Franklin, J., Gottschall, L., Hrovatin, L. A., Parker, S., Doleshal, P., & 
Dodge, C. (2001). A New Approach to Monitoring Exercise Training. The Journal of Strength 
& Conditioning Research, 15(1), 109. 
Hopkins, W. G. (2000). Measures of Reliability in Sports Medicine and Science. Sports Medicine, 
30(1), 1–15. https://doi.org/10.2165/00007256-200030010-00001 
Koo, T. K., & Li, M. Y. (2016). A Guideline of Selecting and Reporting Intraclass Correlation 
Coefficients for Reliability Research. Journal of Chiropractic Medicine, 15(2), 155–163. 
https://doi.org/10.1016/j.jcm.2016.02.012 
McAlinden, C., Khadka, J., & Pesudovs, K. (2015). Precision (repeatability and reproducibility) studies 
and sample-size calculation. Journal of Cataract & Refractive Surgery, 41(12), 2598. 
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project.org/ 
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https://aeitechnologies.com/gas-analyzers/ 
Weir, J. P. (2005). Quantifying Test-retest Reliability Using the Intraclass Correlation Coefficient and 
the SEM. The Journal of Strength & Conditioning Research, 19(1), 231.