The influence of alkalosis on repeated high-intensity exercise performance and acid–base balance recovery in acute moderate hypoxic conditions

Purpose Exacerbated hydrogen cation (H+) production is suggested to be a key determinant of fatigue in acute hypoxic conditions. This study, therefore, investigated the effects of NaHCO3 ingestion on repeated 4 km TT cycling performance and post-exercise acid–base balance recovery in acute moderate hypoxic conditions. Methods Ten male trained cyclists completed four repeats of 2 × 4 km cycling time trials (TT1 and TT2) with 40 min passive recovery, each on different days. Each TT series was preceded by supplementation of one of the 0.2 g kg−1 BM NaHCO3 (SBC2), 0.3 g kg−1 BM NaHCO3 (SBC3), or a taste-matched placebo (0.07 g kg−1 BM sodium chloride; PLA), administered in a randomized order. Supplements were administered at a pre-determined individual time to peak capillary blood bicarbonate concentration ([HCO3−]). Each TT series was also completed in a normobaric hypoxic chamber set at 14.5% FiO2 (~ 3000 m). Results Performance was improved following SBC3 in both TT1 (400.2 ± 24.1 vs. 405.9 ± 26.0 s; p = 0.03) and TT2 (407.2 ± 29.2 vs. 413.2 ± 30.8 s; p = 0.01) compared to PLA, displaying a very likely benefit in each bout. Compared to SBC2, a likely and possible benefit was also observed following SBC3 in TT1 (402.3 ± 26.5 s; p = 0.15) and TT2 (410.3 ± 30.8 s; p = 0.44), respectively. One participant displayed an ergolytic effect following SBC3, likely because of severe gastrointestinal discomfort, as SBC2 still provided ergogenic effects. Conclusion NaHCO3 ingestion improves repeated exercise performance in acute hypoxic conditions, although the optimal dose is likely to be 0.3 g kg−1 BM.


Introduction
Repeated bouts of high-intensity exercise are a frequent feature of training and competition in athletes (Monedero and Donne 2000;Barnett 2006). The recovery between these exercise bouts is an essential component for determining the effectiveness of the subsequent bout. Enhanced recovery can allow athletes to tolerate greater training loads in the subsequent bout, potentially enhancing the post-training adaptation as a result (Barnett 2006). Whereas, in competition, enhancing recovery is an important component to sustain performance within the subsequent bout. This is applicable to sports such as track cycling, swimming, or a rowing regatta series which involve heats, semi-finals, and finals within a short amount of time (Al-Nawaiseh et al. 2016;Monedero and Donne 2000). Specifically, the gap between the men's team pursuit first round and the final at the Rio 2016 Olympics was separated by just 60 min. Considering that most national and Olympic records are achieved within the preliminary rounds of these events (Al-Nawaiseh et al. 2016), this suggests that full recovery is not always possible during these time frames or that current recovery practices are not optimal. Therefore, interventions to improve recovery and sustain subsequent exercise performance are important.
A major factor that may hamper post-training recovery and the subsequent bout of exercise is the metabolic disturbance that occurs following an initial high-intensity exercise bout (Barnett 2006). Ward et al. (2016) reported that the decline in pH and HCO 3 − following a 4 km cycling time trial (TT; team pursuit distance) was substantial, and reflective of metabolic acidosis (pH 7.16 ± 0.08, HCO 3 − 11.9 ± 2.3 mmol l −1 ), which was measured using capillary blood samples. Full recovery of such disturbances is likely to take over 75 min, as Callahan et al. (2017) reported HCO 3 − was 6.2 mmol l −l below baseline at this point (19.5 ± 1.4 vs. 25.7 ± 1.0 mmol l −1 ). If only 60 min is available for recovery, therefore, such as that during track cycling events, an existing acid-base balance perturbation will be evident. Although contentious (Westerblad 2016), critical rises in hydrogen cation (H + ) accumulation are linked to a reduction in both the release and uptake of calcium ions (Ca 2+ ) from the sarcoplasmic reticulum (Allen et al. 2008), disruption of key enzymes of the glycolytic pathway (Hollidge-Horvat et al. 1999), and a reduction in muscle excitability and action potentials by reducing the strong ion difference (SID) (Cairns and Lindinger 2008). In turn, this may hamper subsequent performance by reducing the capability for muscle force production (Cairns 2006;Fitts 2016). It is intuitive to suggest, therefore, that interventions to accelerate post-exercise recovery of acid-base balance could be beneficial for a subsequent bout of exercise.
The ingestion of sodium bicarbonate (NaHCO 3 ) has been shown to accelerate post-exercise acid-base balance recovery and subsequent exercise performance (Pruscino et al. 2008;Zabala et al. 2008Zabala et al. , 2011Gough et al. 2017a). Pruscino et al. (2008) reported a 'trivial' to 'moderate' benefit in the second bout of a 2 × 200 m freestyle swim (interspersed with a 30 min recovery) following pre-exercise ingestion of NaHCO 3 . It was then later reported that NaHCO 3 ingestion 30 min into a 90 min post-exercise recovery significantly improved subsequent cycling capacity at 100% peak mean minute power by 16.6% (Gough et al. 2017a). Both studies reported that capillary blood pH and HCO 3 − recovery were accelerated and above baseline at the end of the recovery period, whereas the placebo condition failed to fully recover. This time frame between bouts might explain why ergogenic effects were observed, as this allowed pH and HCO 3 − to recover sufficiently. Conversely, no effect of NaHCO 3 ingestion has been reported on three repeated Wingate tests separated shorter recovery time frames of between 15 and 30 min (Zabala et al. 2008(Zabala et al. , 2011, or during three repeated high-intensity swimming bouts separated by 20 min (Pierce et al. 1992). Zabala et al. (2008) showed that neither pH, nor HCO 3 − recovered back to baseline levels between the three Wingate tests, suggesting that the recovery of pH and HCO 3 − to this level may be important to produce ergogenic effects in the subsequent exercise bouts.
Despite the promising effects of NaHCO 3 ingestion to improve repeated bouts of exercise in normoxia, this strategy has yet to be applied to acute hypoxia. Nonetheless, Robergs et al. (2005) reported the use of NaHCO 3 combined with sodium citrate lead to post-exercise recovery of pH and HCO 3 − to baseline in approximately 50 min at 1570 m terrestrial altitude, whereas the placebo condition failed to recover to baseline within the 80 min sampling period. Robergs et al. (2005) featured no subsequent bout of exercise, however, and it is also unclear if the participant cohort completed an acclimatisation period in the terrestrial altitude location. This enhanced post-exercise recovery displayed by Robergs et al. (2005) is potentially important, nonetheless, as athletes may complete multiple bouts of high-intensity exercise to maximise the adaptation from hypoxic training schedules, therefore, highlighting the need for optimal recovery. The use of pre-exercise NaHCO 3 ingestion may, in turn, result in a blunting of the initial stress of acid-base balance during an initial bout of exercise, but also improve recovery, leading to improved subsequent exercise performance. This may have a cumulative effect in sustaining training volume and intensity during hypoxic training schedules, particularly considering chronic NaHCO 3 ingestion has been shown to be effective at improving performance following training schedules at sea level (Egger et al. 2014;Durkalec-Michalski et al. 2018). The aim of this study, therefore, was to investigate the effects of both 0.2 g kg −1 BM and 0.3 g kg −1 BM NaHCO 3 on repeated bouts of 4 km TT cycling performance in acute moderate hypoxic conditions. The hypothesis of this study was that both doses of NaHCO 3 would improve both bouts of exercise compared to the placebo; however, 0.3 g kg −1 BM NaHCO 3 would improve performance to the greatest extent.

Participants and compliance with ethical standards
Ten trained male cyclists (age 27 ± 8 years, body mass 82 ± 9 kg, hypoxic maximal rate of oxygen uptake (VO 2max ) 48.5 ± 5.6 ml kg min −1 , and hypoxic peak power output 331 ± 40 W) volunteered for this study. All participants' training load was reflective of a 'trained' cyclist (De Pauw et al. 2013). Ethical approval was granted from the Universities Research Ethics Committee (URESC16-LG01; Edge Hill University), and all participants provided written informed consent.

Experimental overview
Using methods previously described (Gough et al. 2017b), an initial maximal rate of oxygen uptake (VO 2max ) test was conducted in a normobaric hypoxic chamber set at a fraction of inspired oxygen (FiO 2 ) of 14.5% (~ 3000 m). Participants then visited the laboratory on a further six separate occasions in a block randomized, crossover, and double-blind designed study (2 × identification of peak blood HCO 3 − , and 4 × cycling TTs). Individual time to peak HCO 3 − was determined prior to the cycling time trials, using a previously described method (Gough et al. 2017c). This entailed participants ingesting either 0.2 g kg −1 BM NaHCO 3 (SBC2) or 0.3 g kg −1 BM NaHCO 3 (SBC3) on separate occasions, followed by a quiet rest for 180 min. Finger prick capillary blood samples were taken every 10 min, and the highest value of HCO 3 − was then used as the individual time to peak. Twenty-four hours prior to each cycling TT, participants refrained from consumption of alcohol and caffeine, any strenuous activity, and maintenance of nutritional intake, which was confirmed via use of written nutrition/training diaries. Finally, participants were verbally screened to ensure avoidance of beta alanine ingestion had not occurred prior to enrolment onto the study, to account for the long washout of carnosine (Baguet et al. 2009).

Time trial protocol, supplementation of sodium bicarbonate, and blood measures
Participants completed 2 × 4 km TTs (TT 1 and TT 2 ) interspersed with a 40 min recovery. The protocols for the TT, including the self-selected warm-up, were identical to those detailed in the previous research (Gough et al. 2017c), whereby only cadence and gear was displayed to the participant. Each TT series was preceded by supplementation of one of the SBC2, SBC3, or a taste-matched placebo (0.07 g kg −1 BM sodium chloride; PLA) administered in a block-randomized order. The randomisation of supplements was carried out by an individual who was not involved in the research, and performance times remained double-blind until completion of the study. Participants remained seated until their respective pre-determined time to peak HCO 3 − in normoxic conditions. Once reached, participants then entered the normobaric hypoxic chamber (FiO 2 14.5%) for 10 min, prior to beginning the TT 1 warm-up. Following TT 1 , participants completed a passive recovery entailing a quiet seated rest for 40 min within the hypoxic environment. Finger prick capillary blood samples for acid-base balance (pH and HCO 3 − ), electrolytes (K + , Na + , Ca 2+ , and Cl − ), lactate, and haemoglobin saturation with oxygen (SpO 2 ) were recorded pre-exercise, at time to peak HCO 3 − , immediately postexercise and at 10 min intervals during the 40 min passive recovery. These blood samples were immediately analysed using a reliable blood gas analyser (ABL800BA-SIC, Radiometer Medical ltd., Denmark), apart from lactate, which was analysed using a reliable and accurate lactate pro 2 analyser (Arkray, Japan) (Pyne et al. 2000;Bonaventura et al. 2015). To maintain the double-blind nature of the study, the screen on the blood gas analyser was covered, and an individual who was not part of the research stored the data until completion of the study. Calculation of the apparent SID was conducted using the formula ( (Lloyd 2004). Afterwards, participants completed the TT 2 warm-up, followed by TT 2 . Additional blood samples were obtained following the warm-up in TT 2 and immediately post TT 2 for the same measures previously described.

Perceptual measures
Rating of perceived exertion for overall body exertion (RPE O ) and the legs (RPE L ) was recorded during TT 1 and TT 2 every 1 km, whilst HR and SPO 2 were recorded during both TT 1 and TT 2 at every 500 m split and at 10 min intervals during recovery. Gastrointestinal (GI) discomfort was recorded at 10 min intervals after ingestion of the supplement (i.e., SBC2, SBC3, or PLA) up to the individual time to peak HCO 3 − , and at 10 min intervals during the 40 min recovery. At individual time to peak HCO 3 − , participants were asked if they could determine which supplement they had ingested using a supplement belief questionnaire.

Statistical analysis
No evidence of a violation for normality and sphericity was evident in any assessed variable, and therefore, the appropriate parametric statistical tests were employed. A paired t test was conducted for the following: both the time to peak and absolute change in pH and HCO 3 − , and both the severity and aggregated score for GI discomfort following SBC treatments. Performance data (time to TT completion and mean power) and blood parameters (change in pH and HCO 3 − during TT 1 , recovery, and TT 2 ) were analysed using a repeated measures ANOVA. In addition, magnitude-based inferences (MBI) with 90% confidence intervals (CI) were calculated for performance data and interpreted using an adapted method from a freely available spreadsheet (Batterham and Hopkins 2006). The thresholds to depict a benefit or harm were set as the typical error of the 4 km TT when converted from a percentage to an absolute value. This was completed by calculating the difference score in each individual, then calculating the standard deviation of the difference scores, and finally by dividing this by √2 (Swinton et al. 2018). This was used as in many cases, the 0.2 small effect size of Cohen d (Cohen 1988) is less than the typical error and, therefore, produces inflated positive results. Otherwise, a two-way [treatment × time] repeated measures ANOVA was conducted with a Bonferroni correction. Effect size for interactions is reported as partial eta squared (Pη 2 ) and where appropriate, between treatment Hedge's g effect sizes (g) are reported and interpreted as per conventional thresholds (Cohen 1988). Significant effects are displayed with 95% CI where appropriate. Reproducibility of the absolute changes in pH and HCO 3 − in the preliminary trial and the subsequent cycling trials was assessed using intraclass correlation coefficients (ICC). Data are reported as mean ± standard deviation (SD) and statistical significance was set at p < 0.05. Data were analysed using a statistical software package, SPSS (V.22, IBM Inc., Chicago, IL, USA).

Discussion
This study investigated the effects of NaHCO 3 ingestion on post-exercise acid-base balance recovery and repeated 4 km TT performance in moderate acute hypoxic conditions. Both SBC2 and SBC3 improved TT 1 and TT 2 performance compared to PLA, displaying 'likely' and 'very likely' beneficial effects in magnitude-based inferences analysis, respectively. The current study findings suggest that this occurred due to the greater magnitude of acid-base balance recovery between TT 1 and TT 2 or that the initial acid-base balance stress during TT 1 was blunted by NaHCO 3 ingestion. A greater magnitude of performance improvement was observed in SBC3, however, showing 'likely' and 'possibly' beneficial effects in TT 1 and TT 2 compared to SBC2, respectively. As such, SBC3 is the most optimal to improve repeated efforts of high-intensity exercise in acute moderate hypoxic conditions. One individual did display an ergolytic effect following SBC3, however, despite still gaining ergogenic effects from SBC2 vs. PLA, possibly caused by the onset of severe GI discomfort (Saunders et al. 2014). Individuals who display similar responses may, therefore, wish to select SBC2.
The current study findings suggest the enhanced exercise performance in both bouts of exercise following NaHCO 3 ingestion occurred due to the combination of the greater alkalotic state of the acid-base balance prior to TT 1 and the greater magnitude of acid-base balance recovery prior to TT 2 . The change in HCO 3 − during TT 1 was increased following NaHCO 3 (SBC2 + 29%, SBC3 + 34%), whilst post TT 1 , blood lactate was also greater compared to PLA (SBC2 + 21%, SBC3 + 23%). These changes suggest an increased H + buffering from intramuscular to extracellular compartments, which may lead to an increased anaerobic energy provision and glycogen utilization, as intramuscular pH is better protected (Lopes-Silva et al. 2018;Percival et al. 2015). It is argued that these indirect biomarkers of upregulated glycolytic flux in the present study instead show a reduction in lactate by inactive tissue however, as the samples in the present study do provide data on use/ uptake (Granier et al. 1996). Likewise, it is strongly argued that acidosis does not inhibit contractile machinery and that alkalization may cause instability of the buffering systems, and thus, the benefit to the ATP-generating process is either negligible, or actually harmful (Korzeniewski and Zoladz 2002;Sahlin et al. 1998). A recent study by Lopes-Silva et al. (2018), nonetheless, reported similar post-exercise HCO 3 − and lactate responses to the current study following NaHCO 3 ingestion, yet also reported both a 34% greater estimated glycolytic energy contribution to exercise and an improved performance. It is alternately conceivable that the increases in HCO 3 − and CO 2 following NaHCO 3 ingestion are part of a causal sequence of the primary mechanism of performance, by accelerating VO 2 kinetics at the onset of high-power outputs (Zoladz et al. 2005). Unfortunately, VO 2 was not measured during the TTs in the study, so this cannot be concluded and thus warrants further research. The current study findings, nonetheless, further support past research (Fitts 2016) that acid-base balance disturbances are an important determinant of fatigue during high-intensity exercise.
The current study adds that NaHCO 3 ingestion increased the SID prior to each TT bout, which may explain the improved performance. The SID has been suggested to be an important component of action potentials by increasing muscle excitability (Allen et al. 2008). Notable changes Fig. 6 Individual aggregated score and severity of gastrointestinal (GI) responses following SBC treatments. Asterisk denotes SBC3 significantly greater than SBC2 (p < 0.05) NaHCO 3 ingestion elicits include a lowering of extracellular K + and Cl − , whilst increasing Na + , all of which have collectively been identified to be important for muscle contraction during high-intensity exercise previously (Cairns and Lindinger 2008). Here, the current study findings expand upon those of Sostaric et al. (2006) that reported an improvement in finger flexion to exhaustion combined with an increased SID following NaHCO 3 ingestion, by showing a similar effect but during dynamic whole-body exercise. These findings also provide an alternative mechanism to the pH and HCO 3 − -mediated mechanisms often criticised in the literature (Westerblad 2016). It is worth noting that the highlighted changes in the SID may also have further reaching benefits to health-related outcomes. Mild metabolic acidosis has been linked with multiple health-related outcomes including protein metabolism, by decreasing synthesis through increases of non-enzymatic proteolysis (Wiederkehr and Krapf 2001). Likewise, regular consumption of alkaline rich supplements has been shown to improve mineral balance and rate of bone formation (Sebastian et al. 1994). Therefore, more research is required investigating the use of chronic NaHCO 3 ingestion in sporting populations that could also be prone to such issues, particularly endurance runners and adolescent athletes (Tenforde et al. 2017;Scofield and Hecht 2012).
Both pH and HCO 3 − following SBC treatments were increased prior to TT 2 , such that the absolute change in HCO 3 − from TT 1 to 40 min recovery was over twofold greater compared to PLA (SBC2 + 51%, SBC3 + 55%), whilst the change in the SID was also more superior (SBC2 + 29%, SBC3 + 31%). This suggests a greater amount of H + buffering occurred during this time, which subsequently facilitated a more substantial recovery of acid-base balance compared to PLA, in support of previous research (Pruscino et al. 2008;Gough et al. 2017a). Alternatively, the improvement in TT 2 may have been due to the stress on acid-base balance being blunted by NaHCO 3 during TT 1 , as pH, HCO 3 − , and the SID were all greater immediately post TT 1 compared to PLA. Based on data from Gough et al. (2017a), however, it is more likely that the enhancement of the acid-base balance state between TT 1 and TT 2 explains the improvement in the current study, as the authors reported NaHCO 3 ingestion improved a subsequent exercise even when supplementation was after an initial bout of exercise. Moreover, SBC3 elicited a significantly greater magnitude of acid-base balance recovery prior to TT 2 , and change in HCO 3 − during TT 2 compared to SBC2. Correspondingly, SBC3 improved performance within half of the sample compared to SBC2, compared to only two displaying greater improvements in SBC2 vs. SBC3 when using the 3.1 s TE of the test. Combined, these greater increases in acid-base balance blood analytes may explain the greater magnitude of improvement in TT 2 produced by SBC3.
At 40 min recovery pH, HCO 3 − and the SID were still rising following NaHCO 3 ingestion, such that pH at 40 min recovery was 7.49 ± 0.02 in SBC3, which was identical to the increase prior to TT 1 following the same dose (7.49 ± 0.01). This is in agreement with the previous research in normoxia showing similar increases at the end of a recovery period typically seen with pre-exercise NaHCO 3 ingestion (Callaghan et al. 2017;Pruscino et al. 2008). Equally, in the current study, and others (Callaghan et al. 2017;Pruscino et al. 2008), acid-base balance status was still significantly rising to a more alkalotic state. It is plausible to suggest, therefore, if a longer period of recovery was employed, a more pronounced performance effect may have been observed compared to PLA. Moreover, this also suggests that re-dosing NaHCO 3 following an initial fatiguing bout is not required, as acid-base balance increased well above baseline, despite no re-dosing of NaHCO 3 . These findings may be of importance to individuals who suffer from GI discomfort, as no instances were reported during recovery in the current study. Future research should, therefore, investigate the performance responses on repeated exercise following NaHCO 3 ingestion with a longer period of recovery.
One participant presented an ergolytic effect in both TT 1 and TT 2 after ingestion of SBC3. This was likely due to the occurrence of severe GI discomfort (diarrhoea = 10; aggregate score = 63), as this participant still improved their performance in SBC2. These findings support the previous research, whereby ergolytic effects were observed in participants who suffered from severe GI discomfort following 0.3 g kg −1 BM NaHCO 3 (Saunders et al. 2014;Froio de Araujo Dias et al. 2015). It is therefore important to monitor the GI discomfort responses following NaHCO 3 ingestion on an individual basis, as those who display severe symptoms following SBC3 may instead benefit from ingesting SBC2. The use of this smaller dose, however, will be dependent on an improvement in performance still being observed compared to a placebo.

Conclusion
This study investigated the effects of NaHCO 3 ingestion on repeated 4 km TT performance and acid-base balance recovery in acute moderate hypoxic conditions. Both amounts of NaHCO 3 employed in this study ensured recovery of acid-base balance back to baseline or above within 20-40 min, whereas this was not achieved for PLA. For the first time, blunting of the acid-base balance stress during the initial bout of exercise, or a greater magnitude of acid-base balance recovery, has translated into improved subsequent high-intensity exercise performance following NaHCO 3 ingestion in acute hypoxic conditions. The performance improvement was greater in SBC3, which is likely due to the greater alkalotic status of acid-base balance both prior to TT 1 , and during recovery compared to SBC2 and PLA. The onset of GI discomfort was an issue with SBC3, however, and one participant displayed an ergolytic effect on performance following this dose. Individuals should, therefore, employ SBC3 to improve performance in acute hypoxic conditions, only if severe GI discomfort does not occur.
Author contributions Conception or design of the work: the original conception of the work was conducted by LG. Data collection: data were collected by LG. Data analysis and interpretation: completed by LG. Drafting the article: completed by LG. Critical revision of the article: all authors (DB, SD, SAS, and LRM) on the manuscript contributed. Final approval of the version to be published: this was sought from all authors (DB, SD, SAS, and LRM).
Funding No sources of funding were used to conduct this study or prepare this manuscript.