This study was approved by the Midwestern University Institutional Review Board (IRB) prior to subject enrollment. All subjects gave informed consent and understood the procedures, risks, and voluntary nature of the study.

Subjects (ages 21–42) were recruited from Midwestern University in Downers Grove, IL (“controls”: N = 9; 4M, 5F) and a local underwater hockey club (“divers” N = 7; 4M, 3F). All subjects were healthy, non-smokers without apparent cardiovascular or respiratory disease and not taking any medication that would interfere with study parameters. In addition, subjects abstained from caffeine use on the day of testing. No adverse experiences were reported.

A total of eight trials were performed at two lung volumes—four at TLC and four at FRC. Before each breath hold trial, subjects were asked to breathe one of the following four gas mixtures to alter chemoreceptor activity. Standard Air (21% O₂) was the control gas; 100% O₂ was chosen to decrease peripheral chemoreceptor activity; 15% O₂ / 85% N₂ was chosen to increase peripheral chemoreceptor activity; and 5% CO₂ / 95% O₂ was chosen to increase central chemoreceptor activity and decrease peripheral chemoreceptor activity. The assignment of each gas was random, predetermined, and conducted in a single-blind fashion—subjects were blinded to the gas mixture administered before each trial.

Two practice trials were performed prior to recorded testing to acclimate the subjects to the testing format. Four trials with each of the four gas mixtures were conducted at TLC followed by four additional trials at FRC.

The subjects were seated and breathed the appropriate gas mixture for 5 min according to a randomization list, via a disposable facemask. After 3 min. of breathing the gas, an expired CO₂ measurement was obtained by having the subject take a full breath, remove the face mask without releasing any air and blow into an end-tidal CO₂ (etCO₂) analyzer (see below). The subject then replaced the facemask without taking a breath and continued to breathe the gas mixture for an additional 1 min. At the end of the last minute the subject was given a verbal cue to halt their breathing at either TLC or FRC, remove the facemask, and immediately immerse their face in a basin of room temperature water. To ensure full stimulation of the diving response the forehead and eye areas were required to be submerged.

Subjects were instructed to hold their breath as long as comfortably possible, but to end their breath hold after they felt the urge to breathe. They were also instructed to refrain from hyperventilation before the breath hold, to refrain from movements of the chest or other skeletal muscles, including swallowing movements, during the breath hold, and not to release any air into the water. At the end of the breath hold, the subject removed their face from the water and immediately provided a second etCO₂ measurement before taking a breath of room air. They were allowed to breathe normally once this measurement was obtained. Five-minute rest periods separated each trial.

Heart Rate [HR, in beats per minute (BPM)] and arterial blood oxygen saturation (SaO₂, in percent saturation) were recorded throughout each trial using a cardiosync pulse oximeter (Noni^TMn Medical, Model 8700) with toe clip sensor and contact thermal printer printing continuously in real time. The oximeter had a stated accuracy of ± 2% of full scale in the range of 70–100% SaO₂. Systolic and diastolic blood pressure were measured using an automatic digital blood pressure monitor with printer (Omron^®, Model HEM-703CP), and were used to calculate mean arterial blood pressure (BP, in mm Hg). EtCO₂ (in percent) was measured using a CO₂ analyzer (BIOPAC CO₂100C). BHD (in s) was recorded using the touch-print feature of the oximeter. Lastly, an elastic chest band recorded chest excursion to evaluate lung volume and extraneous chest movements during each trial (BIOPAC TSD101B).

Blood pressure was obtained once approximately 30 s prior to the breath hold and once during the breath hold. However, BP readings may not have been obtained during the trials that lasted less than 30 s. EtCO₂ was measured 1 min prior to the breath hold and again immediately after the breath hold was terminated. SaO₂ and HR were measured throughout the 3 min prior to the breath hold until completion of the second CO₂ measurement. Data points were collected at 10-s intervals as well as at the timepoints of breath hold initiation and termination. To minimize recording errors, the subjects were instructed to keep their foot still while the toe clip was in place. Water and air temperatures were recorded prior to the first trial. Finally, all subjects were questioned about adverse experiences.

All data is presented as mean ± standard error (SE). Statistical comparisons were made with a computer statistical package (SigmaStat v14) and p < 0.05 was used as the level of significance. For both the controls and divers, data for males and females were not significantly different, and so were grouped together. For all data, three-way ANOVAs compared controls vs. divers, TLC vs. FRC, and the four gases pre-breathed before the face immersions. Two-way ANOVAs compared TLC vs. FRC and the four gases pre-breathed before the face immersions for HR and SaO₂ at specific time points during the face immersions. One-way repeated measures ANOVAs were used to compare HR and SaO₂ during the face immersion to the control value before the face immersion. Even if the BHD lasted longer than 35 s, statistical analysis on HR and SaO₂ was only done on the first 35 s of the face immersion to facilitate statistical comparisons between groups. If statistical significance was reached during comparisons, it was then followed by post hoc testing (Tukey’s multiple comparison procedure or Dunnett’s multiple comparison vs. control) to determine which values were different from the others. T-tests were used to compare BP and etCO₂ before and after the face immersions.

Like previous studies investigating the human diving response, we found that face immersion in room temperature water produced bradycardia and an increase in blood pressure (Gooden, 1994; Foster and Sheel, 2005; Lindholm and Lundgren, 2009; Fitz-Clarke, 2018). Changing chemoreceptor stimulation by pre-breathing different gases before face immersion had little effect on the development of the bradycardia or the increase in BP. The HR response to breath holding after face immersion was similar at either TLC or FRC. Similar results were found by Andersson and Schagatay (1998) who reported that lung volume [60, 85, or 100% of Vital Capacity (VC)] does not appreciably change HR, skin capillary blood flow or blood pressure responses to breath holding. However, unlike Andersson and Schagatay (1998), we found that there was a greater increase in BP when breath holding at lower lung volumes (FRC) than at higher lung volumes (TLC). The intensification of peripheral vasoconstriction when breath holding at FRC would presumably lead to greater oxygen conservation during the breath hold (Andersson et al., 2000). Instead, however, BHD at FRC was significantly shorter than BHD at TLC.

The physiological breaking point signals the end of the easy-going phase of a voluntary breath hold. When physiological variables build up to the point where a signal to terminate the breath hold has been reached, the breath hold can only be continued through voluntary suppression of the drive to terminate the breath hold (Lin et al., 1974; Hentsch and Ulmer, 1984). The primary physiological variable that is thought to determine the physiological breaking point is an increase in chemoreceptor activity through an increase in PCO₂ or a decrease in PO₂ (Lin et al., 1974; Parkes, 2006). When breath holding with oxygen without face submersion, the physiological breaking point is reached when alveolar PCO₂ reaches 48–54 mm Hg (Lin et al., 1974; Parkes, 2006), although the PCO₂ will be lower if there is simultaneous hypoxia (Lin, 1987).

EtCO₂ increased significantly during face immersions, and we found that breath holds at both TLC and FRC ended at an etCO₂ of about 7%. Andersson and Schagatay (1998) found that breath holds at 60%, 85% and 100% of VC ended at an etCO₂ of approximately 7.4%. When subjects pre-breathed different gases before breath holding, we found no post-immersion etCO₂ at which all face immersions ended. Rather, pre-breathing 100% O₂ or 5% CO₂ / 95% O₂ at FRC, or 5% CO₂ / 95% O₂ at TLC, significantly increased the post-immersion etCO₂. Also, the difference between the pre-immersion etCO₂ and post-immersion etCO₂ was increased after pre-breathing 100% O₂, at least at TLC. These data suggest that there is a synergistic effect between CO₂ central chemoreceptor stimulation and O₂ peripheral chemoreceptor stimulation (Fowler, 1954; Lin et al., 1974). Breathing a high O₂ gas permits breath holds to terminate at higher etCO₂’s. However, there is no absolute etCO₂ that, when reached, terminates a breath hold.

In the present study, BHDs were so short that SaO₂ decreased very little during face immersion. Hemoglobin remained above 98% saturated with oxygen, even after pre-breathing hypoxic gas. Consequently it was unlikely that arterial chemoreceptors were greatly stimulated through increasing hypoxemia during any of these breath holds. Therefore the peripheral chemoreflex, by itself, probably did not contribute to the cardiovascular (i.e., HR and BP) changes observed during the breath holds, or to the attainment of the physiological breaking point. However, even though oxygen saturations did not decrease substantially during the breath holds, PO₂ had a synergistic effect with PCO₂, as evidenced by significant differences in post-immersion etCO₂ after pre-breathing different gases (Figure 4).

Although beginning a breath hold at either TLC or FRC did not appreciably change the cardiovascular responses to breath holding, breath holding at either TLC or FRC did significantly change BHD. BHDs were always longer at TLC than at FRC, even when subjects pre-breathed different gases. Andersson and Schagatay (1998) found that larger lung volumes increased the duration of both the easy-going phase and total breath holding duration. It is thought that a larger lung volume provides a sink that allows a build-up of CO₂ and/or depletion of O₂, thus allowing for a longer BHD (Sterba and Lundgren, 1985, 1988). However, even though there were significant differences in etCO₂’s after pre-breathing different gases, there were no significant differences in BHD for the four face immersions at each of the two lung volumes (Figure 4). This suggests that lung volume, in addition to etCO₂, is an important factor in determining physiological breaking point and breath holding capability.

The effect of lung volume on attainment of the physiological breaking point could stem from slowly adapting pulmonary stretch receptor (SAR) afferent activity. With greater expansion of the lungs with greater lung volumes, SAR activity will increase (Sant’Ambrogio, 1982; Schelegle and Green, 2001). When lung inflation is maintained, SARs characteristically show a long-lasting and sustained discharge (Sant’Ambrogio, 1982; Schelegle and Green, 2001). When breath holding at a large lung volume without face immersion, a Breuer–Hering like reflex may act to prolong maximal breath hold duration (Muxworthy, 1951; Chapin, 1955). We suggest that SAR activity during voluntary breath holding with face immersion at large lung volumes acts within the brainstem central rhythm generator to actively prolong phase II expiration and/or to inhibit generation of inspiration (D’Angelo and Agostoni, 1975; St. John and Zhou, 1990; Sant’Ambrogio and Sant’Ambrogio, 1991). The result of the SAR activity would be to extend the duration of the easy-going phase of a breath hold by suppressing attainment of the physiological breaking point that primarily results from an increase in PCO₂. Thus at large lung volumes, inhibition of breath hold termination would occur due to neuronal interactions within the brainstem central rhythm generator, rather than due to a slower build-up of PCO₂ within the lungs. Additionally, strong involuntary breathing movements are less frequent at high lung volumes, possibly explaining why higher lung volumes allows for longer BHDs (Fitz-Clarke, 2018).

Other explanations of the direct effect lung volume plays on BHD could be that the prolonged absence of SAR activity when breath holding at low lung volumes is, in itself, a respiratory stimulus that leads to the termination of the breath hold and reestablishment of eupnea (Fowler, 1954; Godfrey and Campbell, 1968, 1969). At low lung volumes there would be neither rhythmic nor static SAR afferent activity, because with eupnic breathing, and thus at lung volumes near to FRC, there is normally no SAR activity in humans (Sant’Ambrogio and Sant’Ambrogio, 1991). Additionally, the absence of any SAR input due to the voluntarily initiated apnea may act to terminate a breath hold (Mithoefer, 1959; Delapille et al., 2001). A technique that experienced breath hold divers sometimes use to prolong BHD is to engage in fictive breathing movements by pumping the thoracic cavity while keeping the glottis closed [personal observations; (Lin, 1982)]. This would have the effect of changing lung volume, and thus provide SAR input to the brainstem, even if no fresh air reaches the lungs. Rebreathing during a breath hold extends BHD, even though arterial blood gases do not improve (Fowler, 1954; Godfrey and Campbell, 1968, 1969). Another technique used to prolong breath holds is to open the epiglottis and swallow. Although this would not change lung volume or SAR activity, it would provide a rhythmic input into brainstem respiratory centers. However, swallowing or taking a “false breath” while breath holding reduces the magnitude of diving bradycardia (Gandevia et al., 1978), and involuntary breathing movements during breath holding are thought to be too small to influence the diving response (Andersson and Schagatay, 1998).

The experienced divers in our study were members of a local underwater hockey club. Qualitatively, the diving response of divers was similar to that of controls. However, the BHD of divers was significantly longer than that of controls, although not as long as that seen in experienced underwater hockey or rugby players (Davis et al., 1987; Schagatay and Andersson, 1998), or synchronized swimmers (Oldridge et al., 1978; Bjurström and Schoene, 1987). However, our divers were recreational underwater hockey players, rather than national caliber players as in the study by Davis et al. (1987). Additionally, through our instructions to them, divers ended their face immersions at or near their physiological breaking points, rather than near to their conventional breaking points. During the struggle phase of breath hold diving, the physiological drive to terminate the breath hold is consciously suppressed. Breath holding experience or apnea training can prolong total BHD by delaying the conventional breaking point (Davis et al., 1987; Schagatay et al., 1999, 2000). Thus, with our instructions to our subjects to end their breath holds when they felt the urge to breath and not to use fictive breathing techniques, we negated the training effects that could have given the trained divers a breath holding “advantage”.

We conclude many factors bring about the physiological breaking point during breath holding. These include increased PaCO₂ (as measured by etCO₂), although there is no absolute etCO₂ or change in etCO₂ that, when reached, will initiate the termination of the breath hold. We also conclude that high lung volume, or more specifically the increased pulmonary stretch receptor activity at high lung volume, is an important factor that contributes to the physiological breaking point. We suggest that high lung volume acts centrally to inhibit the termination of breath holds, and thus prolong the duration of the easy-going phase of voluntary breath holding. We also conclude that increased arterial chemoreceptor activity due to the development of hypoxia during the breath hold plays a limited, if any, role in terminating most short duration breath holds. Finally, we conclude that previous breath holding experiences increases breath hold durations without substantially changing cardiorespiratory (HR, BP SaO₂, and etCO₂) responses to face immersion.