All trials were conducted in conformity with the principles of the Declaration of Helsinki. The protocol for group I of the study was reviewed and approved by the research ethics committee at Lund University (LU 25-01), which was the relevant governing authority at the time of trials. The protocol for group II was reviewed and approved by the Swedish Ethical Review Authority (2022-04298), the current governing authority in Sweden. With the recruitment of participants, they were provided written information about, e.g., the procedures, potential risks, and handling of data. At the laboratory, after verbal clarification of test procedures and potential risks involved, the participants provided their oral and written informed consent to participate in this study. Exclusion criteria were age below 18 or above 55 years, any known acute or chronic disease, use of medications (except for contraceptives), and pregnancy. Anomalies in blood pressure, lung function, or electrocardiogram (ECG), measured and evaluated at the beginning of each trial, would lead to discontinuation of the trial.

Healthy volunteers were recruited for the study. The number of recruited participants was based on the expected variance of results, relating to previous, similar studies (Andersson et al., 2008; Perini et al., 2010; Taboni et al., 2019). For group I, eighteen participants volunteered, and another eighteen participants volunteered for group II. Participants for group I were recruited among breath-hold divers (n = 8), under-water rugby players (n = 7), scuba divers (n = 2), or subjects having performed long apneas in previous studies at our laboratory (n = 1), i.e., all participants had some to extensive previous experience of apnea. Of the participants in group II, eleven were either swimmers or breath-hold divers with some to extensive previous experience of apnea, while seven were not performing apnea regularly. The characteristics of both groups are presented in Table 1. Most participants were physically active, and besides diving activities, their self-reported physical training averaged 4 h/week, without a difference between groups. All participants were non-smokers. The participants were instructed to arrive at the laboratory after at least 2 h without any heavy meal or caffeine-containing beverages, with only light physical activity being performed within 12 h of the trial.

When participants arrived at the laboratory [air temperature 21.8°C (0.8), ambient pressure 755 mmHg (10), relative humidity 35.8% (9.8)], they received verbal information about the experimental protocol and the equipment that was to be used. All participants were given the opportunity to ask any questions about the procedures before signing the informed consent form for participation in the study. The participant completed a health questionnaire, after which the height and weight of the participant were measured. Blood pressure was measured in the seated position, while spirometry was performed with the participant in the standing position. Thereafter, the participant assumed a supine position on a mattress, and an ECG was recorded. Blood pressure, lung function, and ECG were assessed for anomalies.

After the ECG had been evaluated and the subject had been cleared for continued participation, the participant assumed a prone position on the mattress, and this position was maintained for the remainder of the test. The participant’s head rested on a removable pillow on top of a container used for cold-water face immersions, and the forearms rested on both sides of the container at the level of the heart (cf. Figure 1 in Schagatay and Andersson, 1998). During the experiments, the water temperature was maintained at 9°C–11°C. The vital capacity in this position was measured, and the volume corresponding to 85% of the vital capacity in the prone position was calculated. The probes of the instruments used for recordings of cardiovascular variables and oxygen saturations (cf. section 2.4) were attached, and the participant was reminded about some of the details in the protocol that was dependent on the participant’s correct performance (e.g., procedures just before and after apneas). The participant was told to relax and avoid voluntary hyperventilation, and to avoid Valsalva and Mueller maneuvers during apneas. After stable cardiovascular data were observed, recordings began and were continuously run until after the end of the last apnea in the protocol.

Group I: From the time recordings began, after 3 min of rest, the subject repeatedly performed apneas either without face immersion or with the face immersed into the cold water, alternating between conditions. The apneas lasted either 15, 30, 45, 60, 90, or 120 s in an order that was unknown to the participant. Each apneic time and condition were performed twice, adding up to a total of 24 apneas. The participant was unaware about the apneic duration that was to be performed and thus had to approach the performance as if each apnea was supposed to be sustained to the individual maximal duration. The apneas were separated by 3-min breathing pauses, during which the participant was breathing through a mouthpiece from an open-circuit spirometry system, with a nose-clip attached during both apnea and eupnea. Apneas were initiated after a countdown from one of the experimenters. During the last 10-s countdown, the participant exhaled to the residual volume through the open-circuit spirometry mouthpiece and inhaled, from a pre-filled rubber bladder, a volume of air equal to 85% of the individual prone vital capacity. During apnea without face immersion, the face was held right above the water surface, whereas during apnea with face immersion, the entire face, including the chin and forehead, was immersed. Without providing any continuous time cues during apnea, the experimenter notified the participant just before the intended end of apnea, so that, on command from the experimenter, the participant could end the apnea with a maximal exhalation through the open-circuit spirometry mouthpiece.

Group II: Each participant in this group performed a total of six apneas. The first two were performed to the individual maximal duration, one of these with and one without face immersion in cold water (alternating the starting order among participants). No time cues were provided by the experimenters during these maximal-duration apneas. Based on the individual maximal duration, an individual submaximal breath-holding time was set for the remaining four apneas; the median difference between individual maximal and sub-maximal apnea times was 16 s. The four sub-maximal apneas also alternated between either apnea with face immersion or apnea without face immersion. During the sub-maximal apneas, the participant was given time cues by the experimenter. The sub-maximal apneas were the ones used for subsequent analysis. All apneas were separated by 5-min breathing pauses, except for the last maximal and first sub-maximal apnea that were separated by a 10-min period. A nose-clip was attached when 30 s remained before apnea, just before the participant began breathing through the open-circuit spirometry mouthpiece. The initiation and end of apneas in group II followed the same procedure as in group I, including inhaling 85% of prone vital capacity from the residual volume to initiate apnea, and ending the apnea with a maximal exhalation through the open-circuit spirometry mouthpiece.

In both group I and group II, beginning 5 minutes after the last apnea, the residual volume in the prone position was measured with a nitrogen-dilution technique (Rahn et al., 1949). In short, after a maximal exhalation to residual volume the participant rebreathed five times through the open-circuit spirometry mouthpiece, which at this point was connected to a rubber bladder initially containing 3 L of 100% O₂. The dilution of inert gas in this closed system, measured in the third exhalation, was used for calculation of the residual volume.

A wall-mounted height measurer and an electric scale (BF214, Omron Healthcare Europe, Hoofddorp, Netherlands) were used to measure height and weight, respectively. Pre-trial blood pressure in the seated, resting position, was measured using an automatic sphygmomanometer (Boso-medicus, Bosch + Sohn GMBH, Jungingen, Germany). A hand-held spirometer (Micro Plus, Micro Medical Ltd., Rochester, England) was used for spirometry measurements in both the standing and prone positions. Because glossopharyngeal insufflation, a technique commonly used by competitive freedivers to increase the volume of air in the lungs, is associated with potential adverse effects (Andersson et al., 2009; Chung et al., 2010; Linér and Andersson, 2010), this technique was not allowed during spirometry or the rest of the protocol. An ECG-monitor (group I: Cardisuny 501, Fukuda ME Kogoyo Co., Tokyo, Japan; group II: Cardiovit AT-1 G2, Schiller, Doral, FL, United States) was used for recording the ECG prior to further testing.

During the trials, respiratory flow and expiratory O₂ and CO₂ fractions were recorded using an open-circuit spirometry system (group I: CPX/D Cardiopulmonary Exercise System, Medical Graphics, Minneapolis, MN, United States; group II: Ergocard Professional, Medisoft, Sorinnes, Belgium). The open-circuit spirometry systems were calibrated using a 3-L syringe (Hans Rudolph, Shawnee, KS, United States) and certified gases (AGA Gas, Lidingö, Sweden, or Linde Gas, Solna, Sweden) prior to the start of each trial. Temperature, barometric pressure, and humidity were measured in the laboratory just prior to each experimental session, and temperature in both the ambient air and the water container used for face immersions were noted just before each apnea. From the recorded expired gas fractions and ambient pressure, end-tidal partial pressures of O₂ and CO₂ (P_ETO₂ and P_ETCO₂) were calculated.

In group I, the HR was recorded continuously with a HR monitor (Polar Vantage NV, Polar Electro Oy, Kempele, Finland). In group II, HR, SV, CO, total peripheral resistance (TPR), and arterial blood pressures were recorded continuously using a finger photoplethysmograph (Finapres NOVA, Finapres Medical Systems BV, Enschede, Netherlands). The Finapres NOVA monitoring system records finger arterial pressure using a finger cuff with a built-in photoplethysmograph (Bogert and van Lieshout, 2005). The finger arterial pressure recording is calibrated using the Physiocal algorithm, and the finger pressure is reconstructed into brachial arterial pressure, applying waveform filtering and level correction, which in turn is calibrated with a brachial blood pressure cuff. The finger cuff and the brachial cuff were unilaterally placed on the left middle finger and over the left brachial artery, respectively. The Finapres NOVA uses the Modelflow® algorithm to calculate cardiovascular variables, such as SV, CO, and TPR, from the recorded finger arterial pressure (Wesseling et al., 1993; Bogert and van Lieshout, 2005). The reconstructed arterial brachial pressure was recorded during the trials.

In group II, the deltoid muscle oxygen saturation (SmO₂) was recorded, every 4 seconds, using a regional oximeter (Nonin SenSmart Model X-100 Universal Oximetry System, Nonin Medical, Plymouth, MN, United States), with an adhesive probe (SenSmart Equanox 8204CA rSO₂ sensor, Nonin Medical, Plymouth, MN, United States) attached to the skin above the left deltoid muscle, 5 cm below the acromion. The arterial hemoglobin oxygen saturation (SaO₂) was recorded continuously using a finger pulse oximeter (Biox 3700e, Ohmeda, Madison, WI, United States), with the probe placed on the left index finger.

The recordings of cardiovascular and respiratory variables began prior to the first test and continued until after the end of the last test using a data acquisition system (MP100, BIOPAC Systems, Goleta, CA, United States) connected to personal computers, and the data was stored for later analysis.

Group I: For each participant, baseline eupneic P_ETO₂ and P_ETCO₂, pulmonary O₂ uptake and CO₂ elimination, as well as HR were calculated as mean values from the period 90–30 s prior to all apneas. P_ETO₂ and P_ETCO₂ were determined from the last expiration before apnea and the first expiration that ended apnea. In addition to the determinations of P_ETO₂ and P_ETCO₂, theoretical partial pressures of O₂ and CO₂ of the pulmonary gas at the beginning of apnea (t = 0) were calculated by combining the composition of the gas of the residual volume and the inhaled ambient air (85% of prone vital capacity). Pulmonary gas exchanges during apneas were calculated from the differences between volumes of O₂ and CO₂ in the lungs at the beginning of and at the end of apnea, representing alveolo-capillary O₂ and CO₂ transfer. The volume of O₂ in the lungs at the beginning of apnea was calculated by adding the volume of O₂ in the rubber bladder to the volume of O₂ in the residual volume. The latter was obtained using the measured end-tidal fraction of O₂ in the last, maximal expiration prior to each apnea. The same calculations were done for the volumes of CO₂ and inert gases. For determination of the lung volume at the end of apnea, it was assumed that the volume of inert gases in the lungs was constant during apnea (Hong et al., 1971; Linér et al., 1993). The O₂ and CO₂ volumes in the lungs at the end of apnea were subsequently calculated using the end-apnea lung volume and the end-tidal fractions of the maximal expiration following each apnea (Andersson et al., 2004). The difference in pulmonary gas volumes between apneas of increasing durations was used to calculate the gas exchange during the different apneic periods, i.e., 0–15, 15–30, 30–45, 45–60, 60–90, and 90–120 s. For determinations of the breath-holding time, the recorded tracings of expiratory O₂ and CO₂ fractions were used (Girardi et al., 2023), and the times required for inhalation from and exhalation to residual volume were included. Apneic values for HR were calculated as means for each of the above specified apneic periods using the 2-min apneas without or with face immersion. For all variables, the different apneic periods’ mean values were compared to the baseline, eupneic value. Also, apneas without and with face immersion were compared.

Group II: For each participant, baseline eupneic mean values for cardiovascular variables (HR, SV, CO, TPR, and arterial blood pressures), SmO₂, and SaO₂ were calculated from the period 90–30 s prior to the sub-maximal duration apneas. The apneic mean values for cardiovascular variables and SmO₂ were calculated from the period 30–120 s into each sub-maximal duration apnea. In addition, for the cardiovascular variables, mean values for the apneic periods 0–15, 15–30, 30–45, 45–60, 60–90, and 90–120 s were calculated. For SaO₂, the nadir in the 0–60 s post-apnea period was determined. P_ETO₂ and P_ETCO₂ were determined from the last expiration before apnea and the first expiration that ended apnea. As in group I, the pulmonary gas exchanges during apneas were calculated from the differences between volumes of O₂ and CO₂ in the lungs at the beginning of and at the end of apneas.

For each participant, individual mean values from the two apneas of each type and duration were calculated. IBM SPSS Statistics, Version 29.0.2.0 (IBM Corp, Armonk, NY) was used to perform statistical analysis. Data was checked for normal distribution, using the Shapiro-Wilk test, before further statistical tests were performed. For analysis of changes during apneas compared to baseline, one-way repeated measures analysis of variance with Bonferroni-corrected pairwise comparisons were used for data following a normal distribution, whereas Friedman tests with Bonferroni-corrected Wilcoxon signed rank test were used for data not following a normal distribution. For analysis of differences between apneas without and apneas with face immersion paired samples, two-tailed t-tests were used for data following a normal distribution, and related-samples Wilcoxon signed ranks tests were used for data not following a normal distribution. Data from participants of group I were compared to data from participants of group II using independent samples, two-tailed t-tests. The level used for accepting significance was p < 0.05. Values reported in the text are means (SD), unless otherwise stated.

All participants completed all apneas to the intended durations. Compared to baseline, the P_ETO₂ was higher and the P_ETCO₂ was lower in the last expiration before apnea (Table 2). The pulmonary PO₂ became even higher and pulmonary PCO₂ even lower with the addition of the large volume of ambient air to the residual volume, i.e., after inhalation of air from the rubber bladder. Thereafter, during apnea, the P_ETO₂ and P_ETCO₂, measured in the first post-apneic expiration, decreased and increased, respectively, with time during both apnea without and with face immersion. For the two longest apneic durations, the P_ETO₂ was lower after apnea without face immersion than after apnea with face immersion (p < 0.01), while the P_ETCO₂ did not differ between apneic conditions.

The baseline, eupneic pulmonary O₂ uptake before apnea was 4.07 (0.69) mL⋅min^-1⋅kg^-1 and the CO₂ elimination was 3.49 (0.90) mL⋅min^-1⋅kg^-1. During apneas, both the O₂ and CO₂ exchanges were increased above baseline for the initial 0–15 s (Figure 1). After this increase, the pulmonary O₂ uptake was gradually reduced so that after 30 s, it was reduced compared to eupneic baseline. The time-averaged O₂ uptake during the entire 2-min apnea without face immersion, 3.57 (0.48) mL⋅min^-1⋅kg^-1, was lower than baseline (p = 0.02 vs. baseline). The O₂ uptake was even further reduced during the entire 2-min apnea with face immersion, to 3.42 (0.48) mL⋅min^-1⋅kg^-1 (p = 0.002 vs. baseline; p = 0.006 vs. apnea without face immersion). During the period 30–120 s into apnea without and with face immersion, when the reduction in gas exchange had stabilized, the O₂ uptake was 71 (14) % and 68 (12) % of baseline O₂ uptake, respectively. After the initial increase, the pulmonary CO₂ elimination displayed a fast reduction from eupneic baseline, being evident after 15 s of apnea, and then stabilizing at these reduced levels. The time-averaged CO₂ elimination was reduced to 1.84 (0.24) and 1.82 (0.25) mL⋅min^-1⋅kg^-1 during the entire 2-min apneas without and with face immersion, respectively (p < 0.001 vs. baseline; NS between apneic conditions).

The baseline, eupneic HR before apnea was 67.6 (10.3) bpm. During apneas, the HR increased above baseline for the initial 0–15 s (Figure 2). Thereafter, the HR gradually decreased, especially during apnea with face immersion, and reached a stable level below the eupneic baseline after 30 s of apnea. During the period 30–120 s into apnea, the HR was reduced from baseline in both apnea without face immersion (p = 0.028 vs. baseline) and apnea with face immersion (p < 0.001 vs. baseline, p < 0.001 between apneic conditions). During this period, the average HR was 94 (13) % and 82 (10) % of baseline HR, respectively.

The mean maximal apnea time was 156 (39) s, with a range among participants of 108–237 s. Based on the individual maximal apnea time, the sub-maximal apnea time was individually determined to be set at an average of 137 (31) s, with a range of 90–195 s. All participants completed all sub-maximal apneas to the intended duration.

Just as in group I, apnea caused a reduction in P_ETO₂, measured in the first post-apneic expiration, in group II (Table 3), and the reduction was greater during apnea without face immersion in cold water (p = 0.010). This was reflected by a lower pulmonary O₂ uptake during apnea with face immersion than during apnea without face immersion (p = 0.012). The P_ETCO₂ was increased by apnea, without a difference between apneic conditions. Likewise, there was no difference in apneic pulmonary CO₂ elimination between apnea with or without face immersion.

The baseline, eupneic HR, SV, and CO before apnea was 71.4 (11.0) bpm, 84.1 (16.5) mL⋅beat^-1, and 6.0 (1.4) L⋅min^-1, respectively. During apneas, the HR of the participants in group II followed a time course that was similar to the time course of the HR of the participants in group I (Figures 3A, D). Starting from a level above baseline for the initial 0–15 s of apnea, the HR gradually decreased, reaching a stable level below the eupneic baseline after 30 s of apnea. During the period 30–120 s into apnea, the HR was reduced from baseline in both apnea without face immersion (90 (10) % of baseline, p < 0.001 vs. baseline) and apnea with face immersion (85 (9) % of baseline, p < 0.001 vs. baseline, p < 0.001 between apneic conditions). Contrary to the HR, the SV never increased above baseline during apneas (Figures 3B, E). Instead, during both apnea without face immersion and apnea with face immersion, the SV was steadily reduced from baseline (p < 0.05 vs. baseline), without a difference between apneic conditions. During the period 30–120 s into apnea, the SV was reduced to 80 (7) % of baseline during apnea without face immersion (p < 0.001 vs. baseline), and to 81 (6) % of baseline during apnea with face immersion (p < 0.001 vs. baseline). With the changes in HR and SV, the CO displayed a time course for changes during apnea that was largely similar to the changes observed for HR (Figures 3C, F). From the baseline level, the CO was gradually reduced for the first 30 s of apnea, after which it remained stable below the baseline level (p < 0.001 vs. baseline). During the period 30–120 s into apnea, the CO was reduced in both apnea without face immersion (73 (11) % of baseline, p < 0.001 vs. baseline) and apnea with face immersion (69 (10) % of baseline, p < 0.001 vs. baseline, p < 0.01 between apneic conditions).

Alongside the cardiac responses to apnea, there were vascular responses. During the baseline period, the TPR was 15.9 (4.5) mmHg⋅min⋅L^-1. From this level, from 15 s into apnea and onwards, the TPR increased (p < 0.05 vs. baseline), during both apnea without and with face immersion (Figures 4A, C). During the period 30–120 s into apnea, the TPR was higher during apnea with face immersion (174 (24) % of baseline, p < 0.001 vs. baseline) than during apnea without face immersion (160 (20) % of baseline, p < 0.001 vs. baseline, p < 0.001 between apneic conditions). The baseline levels for systolic and diastolic blood pressures were 130.4 (13.1) and 75.8 (9.5) mmHg, respectively. With the increases in TPR, there were gradual increases in systolic and diastolic blood pressures during apnea (Figures 4B, D), although the blood pressures were not above the baseline levels for the initial 30 s of apnea. From 30 s into apnea and onwards, there were increases in diastolic blood pressure, during both apnea with and without face immersion, while the increase in systolic blood pressure was not evident until 60 s into apnea. On average during the period 30–120 s into apnea, the diastolic blood pressure was 116 (7) % of baseline during apnea without face immersion (p < 0.001 vs. baseline) and 118 (12) % of baseline during apnea with face immersion (p < 0.001 vs. baseline), without a difference between apneic conditions. During the same period, the systolic blood pressure was 106 (5) % of baseline during apnea without face immersion (p < 0.001 vs. baseline) and 109 (9) % of baseline during apnea with face immersion (p < 0.001 vs. baseline), also without a difference between apneic conditions.

The baseline, eupneic SaO₂ and SmO₂ before apnea was 98.1 (0.8) % and 81.5 (7.1) %, respectively. The SaO₂ was at, or slightly above, the baseline level during the initial 90 s of apnea, after which a gradual decrease in SaO₂ became evident (Figure 5). There was a greater arterial desaturation associated with the apnea without face immersion. On average, the individual nadir SaO₂ was 87.2 (9.5) % after apnea without face immersion (p < 0.001 vs. baseline), and 88.7 (8.2) % after apnea with face immersion (p < 0.001 vs. baseline, p = 0.01 vs. apnea without face immersion). The SmO₂ began to fall immediately upon initiation of apnea (Figure 5). On average during the period 30–120 s into apnea, the SmO₂ was 78.6 (6.4) % during apnea without face immersion (p < 0.001 vs. baseline) and 77.9 (6.4) % during apnea with face immersion (p < 0.001 vs. baseline, p = 0.04 vs. apnea without face immersion).

For variables recorded and analyzed in equivalent ways, the results of the participants of group I and group II were compared. The baseline, eupneic HR did not differ between group I and group II. Also, the relative reduction in HR during the period 30–120 s into apnea did not differ between participants of group I and group II, neither during apnea without face immersion, nor during apnea with face immersion. Likewise, the time-averaged, apneic pulmonary O₂ uptake did not differ between group I and group II. However, the time-averaged, apneic pulmonary CO₂ elimination was slightly lower among the participants of group II compared to the participants of group I (p = 0.02 for apnea without face immersion, p = 0.04 for apnea with face immersion).