Received: 6 June 2020

|

Revised: 5 November 2020

DOI: 10.14814/phy2.14664

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Accepted: 11 November 2020

ORIGINAL RESEARCH

Prior oxygenation, but not chemoreflex responsiveness,
determines breath-hold duration during voluntary apnea
Christina D. Bruce1,2
Trevor A. Day1

| Emily R. Vanden Berg1,3,4 | Jamie R. Pfoh1 | Craig D. Steinback4 |

1

Department of Biology, Faculty of
Science and Technology, Mount Royal
University, Calgary, AB, Canada
2

School of Health and Exercise Sciences,
Centre for Heart, Lung and Vascular
Health, Faculty of Health and Social
Development, University of British
Columbia, Kelowna, BC, Canada
3

Department of Biology, Faculty of
Science, University of Victoria, Victoria,
BC, Canada
4

Faculty of Kinesiology, Sport, and
Recreation, University of Alberta,
Edmonton, AB, Canada
Correspondence
Trevor A. Day, Department of Biology,
Faculty of Science and Technology,
Mount Royal University, 4825 Mt Royal
Gate SW, Calgary, AB, T3E 6K6, Canada
Email: tday@mtroyal.ca
Funding information
Natural Sciences and Engineering
Research Council of Canada, Grant/
Award Number: RGPIN-2016-04915;
NSERC USRA; MRU Internal Research
Grants fund; MRU Distinguished Faculty
Award; University of Victoria

1

|

Abstract
Central and peripheral respiratory chemoreceptors are stimulated during voluntary
breath holding due to chemostimuli (i.e., hypoxia and hypercapnia) accumulating
at the metabolic rate. We hypothesized that voluntary breath-hold duration (BHD)
would be (a) positively related to the initial pressure of inspired oxygen prior to breath
holding, and (b) negatively correlated with respiratory chemoreflex responsiveness. In 16 healthy participants, voluntary breath holds were performed under three
conditions: hyperoxia (following five normal tidal breaths of 100% O2), normoxia
(breathing room air), and hypoxia (following ~30-min of 13.5%–14% inspired O2). In
addition, the hypoxic ventilatory response (HVR) was tested and steady-state chemoreflex drive (SS-CD) was calculated in room air and during steady-state hypoxia.
We found that (a) voluntary BHD was positively related to initial oxygen status in
a dose-dependent fashion, (b) the HVR was not correlated with BHD in any oxygen
condition, and (c) SS-CD magnitude was not correlated with BHD in normoxia or
hypoxia. Although chemoreceptors are likely stimulated during breath holding, they
appear to contribute less to BHD compared to other factors such as volitional drive
or lung volume.
KEYWORDS

breath-hold duration, hypoxic ventilatory response, oxygen, peripheral respiratory chemoreflex,
respiratory chemoreceptors, steady-state chemoreflex drive

IN T RO D U C T IO N

Chemoreflexes play an important role in the respiratory control system by changing ventilation in response to fluctuations
in arterial blood gases located at the bifurcation of the common carotid arteries, and the peripheral chemoreceptors (i.e.,
carotid bodies) detect changes in arterial O2 (PaO2) and CO2
(PaCO2). The peripheral chemoreflex (PCR) increases ventilation in response to rapid decreases in PaO2 (hypoxemia)

and increases in PaCO2 (hypercapnia) in a synergistic fashion
(Fitzgerald & Parks, 1971; Lahiri & DeLaney, 1975; LópezBarneo et al., 2016). The PCR plays a critical role in keeping
arterial blood gases within normal ranges, particularly in the
face of acute or chronic blood gas challenges, such as ventilatory acclimatization in hypobaric hypoxia (Mathew et al., 1983;
Severinghaus et al., 1966). In other populations (e.g., chronic
heart failure), increased PCR sensitivity may contribute to various disease states (Trembach & Zabolotskikh, 2017). Therefore,

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original
work is properly cited.
© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society
Physiological Reports. 2021;9:e14664.		
https://doi.org/10.14814/phy2.14664

wileyonlinelibrary.com/journal/phy2

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assessing PCR sensitivity may be relevant for clinical populations (e.g., sleep apnea) and healthy populations exposed to
acute, chronic, or intermittent bouts of hypoxia (e.g., high altitude trekkers, breath-hold divers).
Common methods used to indirectly assess PCR sensitivity in humans include eliciting transient, respiratory gas tests
in order to measure the resulting ventilatory responses to assess chemoreflex magnitude (e.g., Pfoh et al., 2016; 2017).
The short temporal nature of these tests allow investigators
to assess respiratory responses independent of cardiovascular
responses as well as ventilatory changes elicited from central
chemoreceptor (CCR) stimulation (Pfoh et al., 2016). The hypoxic ventilatory response (HVR) test is one method used to
assess PCR sensitivity to changes in oxygen. The HVR test
can be elicited with acute, transient reductions in the fraction
of inspired oxygen (i.e., FIO2) during poikilo- or isocapnic
conditions (Nielsen & Smith, 1952; Pedersen et al., 1999;
Steinback et al., 2007).
However, the methodology and nature of the HVR test
pose limitations when applied to fieldwork or a clinical setting, requiring cumbersome equipment and potentially provoking respiratory discomfort. Recently, Pfoh et al. (2017)
proposed a new method to assess steady-state chemoreflex
drive (SS-CD). The SS-CD provides an indirect measurement of the ventilatory strategy employed from contributions of both peripheral and CCRs in the steady state, given
the prevailing chemostimuli, including pressure of end-tidal
(PET)CO2 (Torr) and peripheral oxygen saturation (SpO2;
%). Calculating the SS-CD requires only steady-state measures and minimal equipment, which could provide utility in
fieldwork or clinical studies looking to assess chemosensitivity in the context of ventilatory acclimatization (e.g., Bruce
et al., 2018). Voluntary breath-hold duration (BHD) has also
been explored as an alternative test for assessing peripheral
chemoreceptor sensitivity (e.g., Feiner et al., 1995; Trembach
& Zabolotskikh, 2017).
The act of voluntary breath holding (apnea) stimulates the
PCR resulting from concomitant hypoxia and hypercapnia developing at the metabolic rate following the voluntary cessation of breathing. In the untrained person, BHD is determined
by a multitude of factors including (a) initial blood gas concentrations (e.g., oxygen and carbon dioxide), (b) initial lung
volume (e.g., afferent feedback from lung stretch receptors),
(c) metabolic rate, and (d) volitional drive (Skow et al., 2015;
see Parkes, 2006 for review). These factors are difficult to
isolate in humans, but duration of a voluntary apnea is the
most objective measure to use when assessing factors that
contributed to the termination of a voluntary apnea in the
untrained individual (i.e., break point; Lin et al., 1974). Two
specific break points have been identified during a voluntary
breath hold (Lin et al., 1974): the physiological and psychological break point. Physiological break point is identified as
the onset of involuntary diaphragmatic contractions while the

BRUCE et al.

psychological break point is when volitional breath holding
ends and breathing resumes. Physiological break point in individuals with no previous breath-hold training will most often
resume ventilation (psychological break point) from the unfamiliar sensation of an involuntary diaphragmatic contraction.
Intuitively, increased PCR sensitivity would potentially
reduce apneic durations, resulting in a negative relationship
between chemosensitivity and BHD. In fact, carotid body resection (Davidson et al., 1974) and vagal and glossopharyngeal
nerve blockade (Guz et al., 1966) have been found to prolong
BHD. Using mathematical modeling, Goncharov et al. (2017)
concluded that at least 70% of BHD is influenced by chemosensitivity in untrained humans. The HVR test has previously been
shown to predict BHD via multiple linear regression (when accounting for lung volume), whereas the hypercapnic ventilatory
response test (HCVR; in hyperoxia) was not a predictor of BHD
(Feiner et al., 1995). However, Trembach and Zabolotskikh
(2017) found that the duration of an end-inspiratory voluntary
BH was strongly and negatively correlated with PCR CO2 sensitivity when quantified by a single-breath CO2 test.
To what extent chemoreceptor sensitivity, when assessed via
transient gas perturbation tests, predicts BHD in humans remains
somewhat inconsistent in the literature, likely due to variability
in quantifying chemoreceptor sensitivity and the constraints and
confounds of testing neural control of breathing in vivo (Pfoh
et al., 2016). In addition, the extent that SS-CD may be related to
breath holding is currently unknown. We aimed to characterize
the effects of prior oxygenation, HVR, and SS-CD magnitude
on voluntary BHD under conditions of steady-state hypoxia
(13.5%–14% O2), normoxia (i.e., room air), and hyperoxia (five
consecutive breaths of 100% O2) in order to assess the role of
peripheral respiratory chemoreceptor activation on voluntary
BHD. We hypothesized that (a) voluntary BHD would be positively related to the initial oxygen status (i.e., hypoxia, normoxia,
or hyperoxia) prior to breath holding; (b) HVR responsiveness
would be negatively related to BHD in hypoxia, normoxia, and
hyperoxia; and (c) SS-CD magnitude in both normoxia and hypoxia would be negatively correlated with voluntary BHD.

2
2.1

|

M ATERIAL S AND M ETHOD S

| Ethical approval

This study abided by the Canadian Government Tri-Council
policy on research ethics with human participants (TCPS2)
and conformed with the standards set by the latest revision
of the Declaration of Helsinki, except for registration in a
database. Ethical approval was received in advance through
the Mount Royal University Human Research Ethics Board
(Protocol 2015-26a). Following recruitment, all participants
provided voluntary, informed, and written consent to the
study prior to the beginning of the protocol.

   

BRUCE et al.

2.2 | Participant recruitment and
inclusion criteria
A total of 16 participants volunteered for the study (29.9 ± 8.0
years; BMI 23.9 ± 3.5 kg/m2; six males), with only a subset (n = 12) completing the transient HVR test (see Section
2.4, below). Participants were all non-hypertensive with no
reported history of neurological, cardiovascular, or respiratory
illness and were not taking any prescription medications aside
from hormonal birth control. Because the monthly fluctuation
of cycling ovarian hormones (Macnutt et al., 2012) and gender (Pfoh et al., 2016) have been previously shown to be not
related to HVR magnitude, no regard was given to the position in the ovarian cycle in the recruitment of women in this
study. All participants were non-smokers and abstained from
caffeine, alcohol, and exercise for at least 12 hr prior to participation. Following pre-screening and written informed consent,
participants were familiarized with the protocol prior to instrumentation (Figure 1). All data were collected in a quiet and
darkly lit laboratory during mid-day. A subset of these participants were also recruited for a previously published study
(Pfoh et al., 2017), and some HVR and SS-CD values are overlapping in both studies. However, the question of this study
was determined a priori in advance and is independent from
Pfoh et al. (2017), which compared various chemoreflex tests.

2.3

| Instrumentation and data collection

All data were collected using a 16-channel PowerLab system
(Powerlab/16SP ML880; AD Instruments; ADI) and analyzed
offline using commercially available software (LabChart Pro
software 8). Respiratory flow was measured through the use
of a pneumotachometer (HR 800L flow head and spirometer
amplifier; ADI ML141). Instantaneous ventilation (V̇I, L/min)
was determined as the product of breath-by-breath inspired volume (VTI; calculated from the integral of the flow signal) and

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respiratory rate (RR, min−1; calculated by 60 per period of the
flow signal). Breath-by-breath O2 and CO2 were sampled proximal to the mouth and measured in percent using a combined
CO2 and O2 gas analyzer (ADI ML206), calibrated daily. The
pressure of end-tidal (PET)O2 and PETCO2 was calculated and
corrected for BTPS in Torr using the daily atmospheric pressure
(Calgary is ≈1,045 m with PATM of ≈665 mmHg). Calculating
oxygen saturation (ScO2; (%)) was performed using the previously described equation (Severinghaus, 1979) as in previous
studies in our laboratory (Pfoh et al., 2016, 2017) and measured
by a peripheral pulse oximeter (SpO2, ADI ML320) placed on
the index finger of the right hand. ScO2 and SpO2 were observed at all times to ensure values never fell below 70% saturation for safety. For cardiovascular variables, participants were
instrumented for measuring heart rate (HR; ECG electrodes
in lead II configuration using the ADI bioamp ML132) and
beat-by-beat blood pressure (Finometer Pro, Finapres Medical
Systems; calibrated for every subject). Instantaneous HR was
calculated from the R–R interval (60 per period). Mean arterial
pressure (MAP) was calculated as a mean from the raw finger
photoplethysmography arterial pressure tracing. Participants
kept their eyes closed throughout the protocol while listening to
white noise via ear buds to minimize distraction, except when
instructions on breath holding were provided.

2.4

| Hypoxic Ventilatory Response

Following 10 min of quiet sitting for baseline measurements (Table 1), the HVR test was carried out (see Pfoh
et al., 2016, 2017 for detailed methodology and background). The transient HVR test consisted of three consecutive inspired tidal breaths of 100% N2 from a 50 L
Douglas bag. Expired gases were directed to room air once
passing by the gas analyzer port proximal the mouthpiece
of the participant. The transient HVR test was repeated
five times, each separated by 1–2 min to ensure respiratory

F I G U R E 1 Protocol schematic. Following instrumentation, participants breathed room air for 10 min. We then carried out five consecutive
transient hypoxic ventilatory response tests (TT-HVR), comprising three consecutive normal tidal breaths of 100% N2. Following a return to
baseline values, participants carried out two voluntary end-inspiratory breath holds (EI-BH), the first under room air conditions, and the second
following five tidal breaths of 100% O2. Participants then breathed FIO2 0.13.5–014 (13.5%–14%) for ~30 min to each steady state, after which they
performed a third EI-BH in hypoxic conditions

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Variable

Room air (21%
O2)

Hypoxia (13.5%–
14% O2)

Hyperoxia
(5 × 100% O2)

EI-BHD (s)

53.8 ± 16.2**

40.4 ± 13.8**

89.9 ± 38.2**

HR (min−1)

66.7 ± 15.3

73.1 ± 18.0*

—

MAP (mm Hg)

102.1 ± 7.9

99.5 ± 7.3

—

RR (min )

11.3 ± 3.3

12.6 ± 4.0

—

VTI (L)

1.03 ± 0.4

1.1 ± 0.2

—

V̇I (L/min)

10.6 ± 1.5

11.9 ± 1.9*

—

PETCO2 (Torr; BTPS)

33.6 ± 2.1

31.7 ± 1.8*

—

PETO2 (Torr; BTPS)

85.0 ± 3.6

43.3 ± 3.5*

—

ScO2 (%)

96.4 ± 0.4

*

78.6 ± 3.6

—

SpO2 (%)

96.9 ± 0.9

*

78.4 ± 4.0

—

SS-CD (V̇I/SI; a.u.)

30.7 ± 5.2

29.3 ± 4.3

—

HVR (∆V̇I/∆ScO2; L/min/%)

0.38 ± 0.18

—

—

−1

T A B L E 1 Breath-hold duration and
baseline variables in room air and in steadystate normobaric hypoxia

Note: Baseline data obtained while breathing room air and after reaching steady state while breathing
13.5%–14% FIO2 (~30-min).
Abbreviations: and HVR, hypoxic ventilatory response (tested via 5× transient N2 test). EI-BHD, endinspiratory breath-hold duration; HR, heart rate; MAP, mean arterial pressure; PETCO2, partial pressure endtidal carbon dioxide; PETO2, partial pressure end-tidal oxygen; RR, respiratory rate; ScO2, calculated oxygen
saturation; SpO2, peripheral oxygen saturation by pulse oximetry; SS-CD, steady-state chemoreflex drive; V̇I,
ventilation; VTI, inspired tidal volume.
EI-BHD in hyperoxia (third column) is following five consecutive tidal breaths of 100% FIO2. Values are
mean ± SD.
*Statistically different than room air (p < .05);
**Statistically different than all other conditions (p < .001).

gases (PETCO2 and PETO2) and SpO2 levels returned to
baseline. To administer the transient test, an investigator
manually switched the three-way valve between room air
and the 100% N2 gas mixture while monitoring the participants’ respiratory flow signal. Switching between the
inspired gas mixtures was performed during expiration,
largely without the participants’ awareness.

2.5 | Breath holding and steadystate hypoxia
Three separate breath holds were performed following the
HVR protocol. Participants were first coached through two,
randomized, maximal breath-hold maneuvers initiated at the
end of a normal inspiration: one following breathing room
air (normoxia), and another following breathing five normal
tidal breaths of 100% FIO2 from a 50 L Douglas bag (i.e.,
hyperoxia). For these two randomized maneuvers, breath
holds were initiated when all variables returned to baseline
breathing room air (~5–10 min). Following a 10-min break,
the participant was then exposed to a FIO2 of approximately
0.135–0.14 (13.5%–14%), equating to simulated 4,500–
5,000m of altitude. Once the participant reached steady
state (i.e., unchanging variables; e.g., ventilation, PETCO2,
and SpO2; ~30 min), they performed the third breath-hold

maneuver. The initiation of each breath hold began with five
coached breaths at their normal (i.e., resting) tidal volume to
avoid anticipatory changes in breathing rate or tidal volume
(leading to hypocapnia), and each breath hold was held until
volitional break point. No practice breath holds were performed, nor were participants given encouragement throughout the breath hold. Each participant completed testing during
a single laboratory visit (~2 hr).

2.6
2.6.1

| Data Analysis and Statistics
| Baseline Data

For all baseline measures, a mean value over 30 s (i.e., 30-s
bin) that appeared most stable and representative for each
participant's baseline was calculated from a 1-min section
at least 30-s prior to the first HVR test. For cardiovascular
variables, we quantified beat-by-beat HR (min-1) and MAP
(mm Hg). For respiratory variables, we quantified respiratory rate (RR; min−1), breath-by-breath inspired volume (VTI;
L), inspired ventilation (V̇I, L/min; the product of breathby-breath VT and RR), PETCO2 and PETO2 (Torr; BTPS),
and ScO2 (%; calculated from the equation described by
Severinghaus, 1979). ScO2 was also used to avoid the welldescribed delay in measuring peripheral pulse oximetry

   

BRUCE et al.

(Trivedi et al., 1997) and the known differences between
SpO2 and ScO2 in hypoxic conditions (Pfoh & Day, 2016;
Pfoh et al., 2016, 2017). We estimated V̇CO2 from V̇I and
peak fraction of expired (FE)CO2 values in the steady state
in both normoxia and hypoxia (V̇I × FECO2/2), corrected for
STPD (0.813 conversion factor).

2.6.2

| Hypoxic Ventilatory Response

For each trial, ventilatory responses were calculated as a
change in ventilation from baseline values to the peak responses (ΔV̇I) over a change in stimulus (ΔScO2). The ΔV̇I
was calculated as the difference (delta) between baseline V̇I
prior to the test (15 s bin) and as the average V̇I of the two
largest consecutive breaths within the first 20 s following the
stimulus, wherever it occurred, similar to previous studies
in our laboratory (Pfoh et al., 2016; 2017). The ScO2 stimulus was calculated using the visually identified PETO2 on the
third 100% N2 breath. The average response of five trials was
taken as the representative value for that participant.

2.6.3

| Breath Holding

Using respiratory flow in conjunction with inspired and expired volume measurements, BHD was determined using
LabChart. Although we did not attempt to measure the physiological breakpoint (e.g., respiratory effort belt), with participants being untrained in breath holding, their final break
point most likely represented both psychological and physiological break point (Parkes, 2006).

2.6.4

| Calculating SS-CD

During baseline periods while breathing room air and during steady-state hypoxia, a stimulus-index was calculated
(SI; PETCO2/SpO2; Torr/%; e.g., Bruce et al., 2016; Pfoh
et al., 2017) to represent the contributions from CO2 and O2
chemostimuli acting on both central and peripheral chemoreceptors (i.e., PETCO2 and SpO2). The SI was then indexed
against baseline V̇I during both normoxic and hypoxic
steady-state conditions to calculate SS-CD (Pfoh et al., 2017;
see Table 1).

2.7

| Statistics

All values are reported in Table 1 and Results as mean ± standard deviation (SD). Statistical significance was assumed
when p < .05 (SigmaPlot v14, Systat).

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Average baseline values during room air and steady-state
hypoxia are summarized in Table 1 for all participants. Paired
t-tests were used to test for differences in variables between
normoxia and hypoxia.
In order to assess the statistical relationship between
apneic duration and initial oxygen status, a one-factor repeated-measures analysis of variance (ANOVA) test was
performed (Table 1 and Figure 2a). When significant Fratios were detected, a Student–Newman–Keuls post hoc
test was used for multiple pair-wise comparisons between
the three oxygen levels (hypoxia, normoxia, and hyperoxia;
Figure 2a).
Paired t-tests were used to compare the delta (Figure 2b)
and percent change (Figure 2c) in BHD from normoxia between hypoxia and hyperoxia.
Pearson product moment correlation tests were used
to assess relationships between (a) all absolute breath hold
durations (BHD) in hypoxia, normoxia, and hyperoxia
(Figure 3a–c) and the HVR; (b) the delta (Figure 4a,b) and
percent change (Figure 4c,d in BHD from normoxia and the
HVR in hypoxia (Figure 4a,c) and hyperoxia (Figure 4b,d);
and (c) the relationship between BHD in normoxia and hypoxia and the respective SS-CD.
Lastly, a paired t-test was also used to compare SS-CD
between normoxia and hyperoxia.

3

|

RESULTS

3.1 | Cardiorespiratory Variables in
Normoxia versus Hypoxia
Table 1 presents baseline cardiorespiratory data while breathing
room air (normoxia) and after reaching steady state (~30 min)
while breathing steady-state hypoxia (FIO2 0.135–0.14). Of note,
HR, V̇I, PETCO2, PETO2, ScO2, and SpO2 were all statistically
different in hypoxia compared to normoxia. Estimated V̇CO2
was marginally, but significantly, higher in steady-state hypoxia
compared to room air (249.3 ± 46.5 versus 232.8 ± 32.8 ml/min,
respectively; +7.0 ± 12.9% increase in hypoxia; p = .03).

3.2 | Normoxic, hyperoxic, and
hypoxic BHDs
For the normoxic breath hold (i.e., breathing room air), participants held their breath, on average for 53.8 ± 16.2 s. Hyperoxic
BHDs were longer than normoxia at 89.9 ± 38.2 s (p < .001).
Breath holds following steady-state hypoxia were statistically
shorter than normoxia at 40.4 ± 13.8 s (p < .001; see Figure 2a).
In addition, compared to room air (i.e., normoxia), the steadystate hypoxic breath hold was −13.4 ± 8.7 s (−24.5 ± 3.6%)

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F I G U R E 2 Breath-hold duration (BHD) in steady-state hypoxia, normoxia, and hyperoxia. (a) Absolute BHD in steady-state hypoxia (13.5%–
14% O2; 40.4 ± 13.8 s), normoxia (room air; mean 53.8 ± 16.2 s), and hyperoxia (following five tidal breaths of inspired 100% O2; 89.9 ± 38.2 s).
(b) Changes in BHD from normoxia in steady-state hypoxia (−13.4 ± 8.7 s) and hyperoxia (36.1 ± 26.1). (c) Percent changes in BHD from
normoxia in steady-state hypoxia (−24.5 ± 14.5%) and hyperoxia (65.1 ± 37.7%). Each transparent circle represents one individual, and horizontal
bars represent the mean. *Significantly different from normoxic breath hold (p < .001)

F I G U R E 3 Within-individual correlations between absolute BHD and the hypoxic ventilatory response (HVR). (a) Relationship between
Absolute BHD in steady-state hypoxia (13.5%–14% O2) and HVR (r = .45, p = .14, n = 12). (b) Relationship between absolute BHD in normoxia
(i.e., room air) and HVR (r = .54, p = .07, n = 12). (c) Relationship between Absolute BHD in hyperoxia (following five breaths of 100% O2) and
HVR (r = .46, p = .13, n = 12). The respective r value (Pearson correlation coefficient), p value, and n are presented on each graph

shorter, while the hyperoxic breath hold was + 36.1 ± 26.1 s
(+65.1 ± 37.7%) longer (Figure 2b,c).

3.3

| HVR and SS-CD

Using the transient 100% N2 test, the HVR was on average
0.38 ± 0.18 L/min/% ScO2 (n = 12; Table 1), similar to previous reports in our laboratory (Pfoh et al., 2016, 2017). SS-CD
was not different between normoxia (30.7 ± 5.2) and hypoxia
(29.3 ± 4.3; p = .2; n = 16; Figure 5a), similar to previous reports in our laboratory (Bruce et al., 2018; Pfoh et al., 2017).

3.4

| BHD and the HVR

Both absolute (delta) and relative (% change from normoxia) BHDs appeared to have no significant relationship with HVR magnitude. BHD in hypoxia (Figure 3a),
normoxia (Figure 3b), and hyperoxia (Figure 3c) was not
related to the HVR (r < .54; p > .07, n = 12). The change
in BHD from normoxia to hypoxia was not correlated with

HVR (r = −.27, p = .39, n = 12; Figure 4a), or was the
change in BHD from normoxia to hyperoxia (r = 0.33,
p = .3 n = 12; Figure 4b). Similarly, percent change in
BHD from normoxia to hypoxia was not correlated with
HVR (r = .002, p = .995, n = 12; Figure 4c), or was the percent change in BHD from normoxia to hyperoxia (r = .06,
p = .84, n = 12; Figure 4d).

| BHD and SS-CD

3.5

There were no differences in SS-CD between normoxia and
hypoxia (p = .2; Figure 5a). Voluntary BHD was not correlated
with SS-CD in either normoxia (r = −.05, p = .85, n = 16;
Figure 5b) or hypoxia (r = .11, p = .69, n = 16; Figure 5c).

4

|

DISCUSSION

We aimed to assess the relationship between prior oxygenation on voluntary BHD, and to assess the possible relationship among BHD, HVR, and SS-CD, within individuals. The

   

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F I G U R E 4 Within-individual correlations between relative BHD and the hypoxic ventilatory response (HVR). (a) Relationship between delta
BHD from normoxia in steady-state hypoxia (13.5%–14% O2) and HVR (r = −.27, p = .39, n = 12). (b) Relationship between delta BHD from
normoxia in hyperoxia (following five breaths of 100% O2) and HVR (r = .33, p = .3, n = 12). (c) Relationship between percent change in BHD
from normoxia in steady-state hypoxia and HVR (r = .002, p = .995, n = 12). (d) Relationship between percent change BHD from normoxia in
hyperoxia and HVR (r = .06, p = .84, n = 12). The respective r value (Pearson correlation coefficient), P value, and n are presented on each graph

principal findings of our study are as follows: (a) BHD duration was dependent on the initial fraction of inspired oxygen
immediately before voluntary apnea, such that BHD is positively related to prior oxygenation in a dose-dependent fashion
(i.e., shorter in hypoxia and longer in hyperoxia); (b) hypoxic,
normoxic, and hyperoxic BHD were not related to the HVR;
and (c) normoxic and hypoxic BHD were not correlated with
SS-CD, which takes into account both central and peripheral
chemoreceptor contributions to BHD in the steady state.

4.1

| BHD and Prior Oxygenation

We found that alterations in prior FIO2 affected voluntary
BHD, indicating peripheral chemoreceptor activation (hypoxia; i.e., decreased duration) and inhibition/withdrawal
(hyperoxia; increased duration) plays an important role in

determining BHD. These results are consistent with previous
studies, which assessed the effects of initial oxygen status on
voluntary BHD (e.g., Engel et al., 1946; Ferris et al., 1946;
Godfrey & Campbell, 1969; Klocke & Rahn, 1959). For example, Engel et al. (1946) found inspired oxygen tensions (below
that of sea level values) prior to initiating a breath-hold drastically decreased duration, which was less pronounced when
approached prior inspired oxygen tensions of 100%. Although
our results do not suggest an exponential relationship per se
between prior oxygen tension and BHD, our data still indicate
BHD is somewhat dependant on initial inspired oxygen.

4.2

| BHD and Chemoreflex Magnitude

Breath holding is a unique stressor, eliciting incremental hypoxia, hypercapnia, and sympathetic nervous

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F I G U R E 5 Within-individual correlations between relative BHD and steady-state chemoreflex drive (SS-CD). (a) Comparison of SS-CD in
normoxia (room air) and hypoxia (13.5%–14% O2). (b) Correlation between absolute BHD and SS-CD in normoxia (r = −.05, p = .85, n = 16).
(c) Correlation between absolute BHD and SS-CD in hypoxia (r = .11, p = .69, n = 16). NSD, no significant difference (p > .05). The respective r
value (Pearson correlation coefficient), p value, and n are presented on each graph

system activation (Hagbarth & Vallbo, 1968; Lin et al., 1974;
Steinback et al., 2009). These concomitant blood gas chemostimuli accumulate at the metabolic rate, stimulate both
central and peripheral chemoreceptors, which continuously
increases the drive to breathe and likely reduces BHD. Thus,
chemoreceptor stimulation or withdrawal is contributing to
the timing of break point during a voluntary breath hold.
However, it is interesting that BHD did not correlate with
HVR magnitude using a transient HVR test, which quantifies the PCR sensitivity to transient reduction in arterial hypoxia. Thus, although chemoreceptor stimulation influences
BHD, chemoreflex magnitude does not appear to determine
voluntary BHD. The lack of correlation between voluntary
apnea duration and chemoreceptor sensitivity measured as
HVR may be a result of (a) the known variability inherent in
transient gas perturbation tests (Pfoh et al., 2016; 2017), (b)
the prevailing CO2 at the beginning of and throughout the BH
and the concomitant sensitivity of the carotid bodies to CO2
(e.g., Trembach & Zabolotskikh, 2017), and (c) the contribution of central respiratory chemoreceptors.
Few studies have assessed the relationship between chemoreflex magnitude and BHD. Bain et al. (2017) showed
that forced vital capacity (an index of lung volume) was a
good predictor of BHD following administration of hyperoxia in elite apneists, but that the central chemoreflex magnitude, assessed via hyperoxic rebreathing tests prior to the
apnea, was not predictive of duration. Feiner et al. (1995)
showed that the HVR was a significant predictor of BHD,
but only when the effect of lung volume was included in statistical analysis. In addition, Feiner et al. (1995) also found
no relationship between voluntary BHD and the central hypercapnic ventilatory response. Conversely, Trembach and
Zabolotskikh (2017) found that PCR magnitude to CO2,
tested via single breath test, was strongly, significantly, and
inversely correlated with voluntary BHD in a large group of
healthy participants. This is difficult to reconcile with the
findings from our study, where the HVR not significantly
correlated with BHD in normoxia or hypoxia, particularly

given that we found previously that the PCR magnitude of
HVR via transient N2 test was well correlated with the PCR
magnitude tested via the single-breath CO2 test, within individuals (Borle et al., 2017), which was also consistent with
a previous study (Rebuck et al. (1973). Thus, an integrative
model of the underlying physiological and extra-physiological mechanisms determining volitional break point remains
to be determined.
Because voluntary apnea elicits a simultaneous blood
gas, circulatory and neural responses that increase cerebral perfusion (e.g., Steinback & Poulin, 2008; Steinback
et al., 2009; Willie et al., 2012), while limiting perfusion of
non-vital organs (i.e., the mammalian diving reflex; Dujic
& Breskovic, 2012; Dujic et al., 2013; Joulia et al., 2009;
Lin, 1982), the CCRs may have played a smaller role in determining break point compared to the PCRs. This mechanism would theoretically dampen the CO2/H+ stimulus to the
CCRs by increasing cerebral blood flow and, thus, promoting metabolic washout from cerebral tissues (e.g., Ainslie &
Duffin, 2009; Bruce et al., 2016). Although cerebrovascular
reactivity (CVR) likely dampens the role of CCR stimulation on determining BHD, this conclusion is only speculation without assessing a relationship between CCR and CVR
during BHD.

4.3

| Possible Effects of CO2/[H+]

The incremental increase in CO2 during breath holding likely
stimulates the central respiratory chemoreceptors, increasing
the drive to breathe and likely shortening BHD (Ferris et al.,
1946). The concomitant hypercapnia during breath holding
also plays a role in stimulating PCRs, particularly as oxygen levels decrease during apnea, given that the PCRs detect
changes in oxygen in a multiplicative, CO2/[H+]-dependent
fashion (e.g., Fitzgerald & Parks, 1971; Kiwull-Schone
et al., 1976; Lahiri & DeLaney, 1975; Lahiri et al., 1978; Van
Beek et al., 1983). In our study, these increases in CO2 may

BRUCE et al.

confound our results in two possible ways: (a) the metabolic
rate (i.e., CO2 accumulation) may have been different during
the three BH trials, (b) hypoxia-induced lactic acid accumulation may alter hypoxic sensitivity, and (c) the baseline (i.e.,
prior to breath holding) PETCO2 may have been different
between conditions. The mean metabolic rate (i.e., V̇CO2)
was only slightly (but significantly) higher (~7%) after 30min hypoxia, which likely played a limited role in increasing CO2 during breath holding under hypoxic conditions. In
addition, 30 min of hypoxia may have generated increased
blood lactate, contributing to arterial acidosis, which we did
not measure. However, Ainslie et al. (2014) showed that following 15-min steady-state hypoxia at levels similar to our
study (PETO2 ~ 42 Torr, SaO2 ~ 78%), arterial lactate was not
increased statistically, however, it was significantly elevated
at more extreme hypoxia (PaO2 ~ 36 Torr, SaO2 ~ 70%; 0.6
to 0.7 mmol/L). With respect to more chronic hypoxia, Smith
et al. (2014) showed that on days 4–6 at 5,050 m following
9 days of incremental ascent (PaO2 ~ 42 Torr, SaO2 ~ 81%),
arterial lactate was only mildly but statistically elevated (0.7
at baseline to 0.9 mmol/L). Conversely, baseline PETCO2
was lower in hypoxia by approximately 2 Torr (see Table 1),
which was caused by and subsequently inhibited the HVR
(i.e., poikilocapnic hypoxia; e.g., Steinback & Poulin, 2007;
see Table 1). Indeed, ventilation was statistically higher at
baseline after 30 min of hypoxia, but only by approximately
1 L/min on average. Thus, participants had higher ventilation
in hypoxia, but it was blunted by the relative concomitant hypocapnia. These two antagonistic factors, where V̇CO2 was
higher but starting CO2 was lower compared to the normoxic
BH, (a) are likely negligible in their contribution to BHD and
(b) likely cancel each other out across the breath hold, leaving the effects of prior oxygenation as the key variable driving differences in BHD in our study. Indeed, this complex
interplay in generating a new steady state in the face of antagonistic stimuli was in part the initial justification for developing and applying our SS-CD metric (see Bruce et al., 2018;
Leacy et al., 2020; Pfoh et al., 2017).

4.4

| BHD and SS-CD

We developed a metric of SS-CD, which encompasses the
ventilatory strategy employed in response to the prevailing
CO2 and O2 in the steady state (Pfoh et al., 2017). SS-CD
was not different between room air and steady-state hypoxia,
likely due to the fact that PETCO2 and SpO2 both decreased
a similar magnitude, while ventilation was only minimally
increased (~1 L/min) compared to room air values once
steady state was reached in hypoxia (see Table 1). Our results suggest that the SS-CD does not determine BHD when
compared in room air and normobaric steady-state hypoxia.
Although we found no correlations between BHD and SS-CD

   

| 9 of 12

magnitude in either acute normoxia or hypoxia, there may be
merit in assessing the relationship between BHD and SS-CD
during chronic hypoxic conditions (i.e., high altitude), where
hypobaric hypoxia decreases voluntary breath hold duration
(e.g., Ferris et al., 1946) and ventilatory acclimatization increases SS-CD (e.g., Bruce et al., 2018; Leacy et al., 2020).
However, it is possible that the temporal domain of breath
holding (i.e., acute dynamic chemostimulation) and calculating the SS-CD (i.e., obtained during steady state) may be
unrelated. In other words, ventilatory chemoresponses to dynamic and incremental changes in blood gases (both CO2 and
O2) may be more relevant to driving voluntary BHD.

4.5 | Potential Contribution of ExtraChemoreceptor Factors
Despite the clear contribution of initial oxygen status on voluntary BHD demonstrated by this and other studies, neither
the transient HVR nor SS-CD was significantly correlated
with BHD. Thus, despite stimulation of central and peripheral respiratory chemoreceptors before and during the breath
hold, chemoreflex magnitude does not appear to determine
voluntary BHD, implicating extra-chemoreceptor factors.
Previous studies demonstrated that sensitivity of the carotid
body is not solely responsible for apnea break point, as individuals following carotid body denervation still voluntarily terminated a breath hold (Davidson et al., 1974). In one
of the most striking examples of extra-chemoreceptor contributions, Campbell et al. (1966), Campbell et al. (1967),
Campbell et al. (1969), showed that voluntary BHD was increased by two to threefold in two participants following full
paralyzation (aside from an upper arm), where participants
indicated their volitional break point with a hand signal from
the unanesthetized hand. These authors suggested that a portion of the distressing sensations elicited by breath holding
arise through the elimination of respiratory muscle contraction and related inhibitory afferent feedback on the drive to
breath (Campbell et al., 1967). Indeed, Fowler, 1954; later
confirmed by Flume et al., 1994) showed that if participants
rebreathed from a bag containing hypoxic and hypercapnic
air at break point, they could continue breath holding, despite
no change in blood gases, illustrating that the act of breathing
itself relieves the symptoms of distress associated with breath
holding, at least initially. However, as the successive break
point–rebreathing cycle continues, the BHD shortens, illustrating a role for chemoreceptor stimulation in the sensation
of breathlessness and voluntary BHD.
In addition to prior oxygenation and afferent feedback from contracting respiratory muscles, other factors
contribute to BHD, such as the initial lung volume (e.g.,
Feiner et al., 1995; Godfrey & Campbell, 1968; 1969; Guz
et al., 1966) and volitional factors (Parkes, 2006). In our

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|   

study, we coached participants to initiate the breath hold at
the end of a normal inspiration, where lung volume at the
onset of each apnea was not equivalent between conditions
(e.g., smaller tidal volumes during normoxia compared to
hypoxia; see Table 1). However, given the difference between measured tidal volume was ~ 100ml between normoxia and hypoxia, these differences likely contributed
little to BHD in comparison to the measured differences in
FIO2. Lastly, performing an additional motor or cognitive
task (Alpher et al., 1986) as well as the simple act of training (Engan et al., 2013) can prolong BHD. The potential
effects of training and volition were controlled for in our
study by selecting untrained participants, and providing no
encouragement to the participants throughout their apnea.
Unfortunately, the effect of training somewhat difficult to
control for when multiple breath holds are required for data
collection, as participants can often breath hold for longer durations with subsequent apneas in the same session
(unpublished observations). Taken together, these considerations demonstrate that breathing holding is a complex
physiological stressor, with multiple factors contribution to
the ability of participants to voluntarily resist one of our
most primitive and immediate physiological drives (e.g.,
Parkes, 2006).

4.6

| Possible Relationship to Sleep Apnea

Central and obstructive sleep apnea are comprised of repeated cycles with alternating phases of breath holding and
hyperventilation during sleep (Douglas et al., 1982; Sin
et al., 2015). Previous experimental and modeling studies
demonstrated a relationship between chemoreflex responsiveness (i.e., gain) and the severity of central sleep apnea,
particularly with ventilatory acclimatization to high altitude
(Ainslie et al., 2013). Because of the differences in temporal delay in stimulating peripheral (short) and central (long)
chemoreceptors (e.g., Pederson et al., 1999), it is likely that
peripheral chemoreceptors play a more important role in
determining the apnea–hyperventilation cycle lengths. In
addition, the transient chemoreflex tests utilized to assess
HVR or HCVR are similar to the kind of chemostimuation
sleep apnea patients’ experience (i.e., short duration chemostimulation). However, perhaps combining transient hypoxia and hypercapnia into a single transient test may be a
more relevant “real world” stimulus. Conversely, although a
strength of the SS-CD metric is that it takes into account the
ventilatory strategy employed in response to the prevailing
CO2 and O2 in the steady state, that this metric is not in fact
a measure of gain (responsiveness) to a perturbation in the
way the hyperventilation following an apneic event is, may
explain the lack of a relationship between SS-CD magnitude and BHD in either normoxia and hypoxia. Using the

BRUCE et al.

appropriate test to assess chemoreflex gain when assessing
its relationship to the severity of sleep apnea remains an important and evolving question (e.g., Messineo et al., 2018;
Solin et al., 2000).

5

|

CONCLUSION

We assessed the relationship between varying levels of inspired oxygen and voluntary BHD in a laboratory setting,
and assessed the possible relationships between both prior
inspired oxygen (i.e., hypoxia, normoxia, and hyperoxia) and
SS-CD on voluntary BHD. We found that BHD was positively related to prior oxygen status, shorter in hypoxia and
longer in hyperoxia. However, BHD was not related to (a)
transient HVR or (b) SS-CD, a metric combining all chemostimuli in the steady state. We conclude that voluntary BHD
is oxygen dependent, likely through PCR activation, but that
extra-chemoreflex factors determine BHD in untrained individuals, such as lung volume, lung stretch, and volition.
ACKNOWLEDGEMENTS
We wish to gratefully acknowledge the contribution of our
participants for their time.
CONFLICT OF INTEREST
None declared.
AUTHOR CONTRIBUTIONS
TAD, ethics, funding, and laboratory where all experiments
took place, conception and design of the work; TAD, JRP,
CDB, EV, and CDS, data acquisition, analysis, and/or interpretation of data for the work; All co-authors were involved
in drafting and/or critically revising the work for important
intellectual content.
FUNDING INFORMATION
Financial support for this work was provided by (a) an NSERC
USRA (C.D.B.), (b) an MRU Internal Research Grants fund
(J.R.P.), (c) an MRU Distinguished Faculty Award fund
(J.R.P.), (d) a University of Victoria Co-op (E.R.V.), and
(e) a Natural Science and Engineering Research Council of
Canada Discovery grant (TAD; RGPIN-2016-04915).
ORCID
Trevor A. Day

https://orcid.org/0000-0001-7102-4235

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How to cite this article: Bruce CD, Vanden Berg ER,
Pfoh JR, Steinback CD, Day TA. Prior oxygenation,
but not chemoreflex responsiveness, determines
breath-hold duration during voluntary apnea. Physiol
Rep. 2021;9:e14664. https://doi.org/10.14814/​
phy2.14664