Forty-two untrained, but recreationally active individuals (27 males, 15 females) age 21 ± 1 years, body mass 72.1 ± 11.4 kg, and height 173 ± 10 cm volunteered to take part in this experiment (Table 1). No differences in anthropometric or fitness measures were found between groups (p > 0.05). Participants were informed of the procedures to be employed in the study and associated risks, which had the approval of the University of Brighton Research Ethics Committee (ESREGC/06/14). All participants provided written, informed consent. The participants were non-smokers and had not spent time above 2000 m in the 2 months prior to the study. Participants were advised to refrain from alcohol and caffeine for 24 h prior to testing and to maintain their normal unstructured training habits (<2 hr.wk) throughout the study.

The 42 participants were randomly assigned and equally split for number (n = 14) and sex (9 males, 5 females), to one of the three intervention groups; a normoxic (NORM) (FiO₂: 0.2093) environment, a moderate normobaric hypoxic (HYP) (FiO₂: 0.15, range 0.148–0.152; FiCO₂: 0.0008, range 0.0003–0.0028) environment and a control (CONT) normoxic non-training group (Table 1). All testing was performed in a nitrogen enriched normobaric hypoxic chamber with temperature (19°C) and humidity (40%) regulated by air conditioning (Altitude Centre, London, UK).

Familiarization of the Wingate anaerobic test (WAnT) and time to exhaustion (TTE) was performed prior to any of the experimental testing. Preliminary testing involved participants completing in sequence, a peak oxygen consumption (V˙O2peak) incremental test, a time to exhaustion cycle test (TTE) and a Wingate anaerobic test (WAnT), with 24 h separating each test. Prior to each V˙O2peak test, venous blood was taken to measure haematocrit (Hct), hemoglobin (Hb), Interleukin- 6 (IL-6), and TNFα, see Figure 1. The SIT consisted of six WAnT sessions over a 2-week period with 24–48 h between each session (Figure 1). Each training session followed that an established SIT protocol (Burgomaster et al., 2005), consisting of an increasing number of WAnTs [four to seven 30 s “all out” efforts on a cycle ergometer interspersed with 4 min warm up/recovery (60 W)]. Throughout training heart rate [HR, bts.min⁻¹ (Polar FT1, Polar Electro, Kempele, Finland)], peripheral arterial oxygen saturation [SpO_2, % (PalmSat 2500, Nonin Medical Inc., Minnesota, USA)], and rating of perceived exertion [RPE; Borg Scale 6–20 (Borg, 1982)] were measured immediately after each WAnT and every minute thereafter during recovery. Those in the CONT group maintained usual physical activity regimes for the 2 week period. Forty-eight hours after the final SIT session all participants repeated the V˙O2peak, TTE and WAnT protocols, each separated by 24 h.

Participants performed an incremental test to volitional exhaustion on an electromagnetically-braked cycle (Schoberer Rad Messtechnik equipped with 8 strain gauges, SRM, Germany), with the zero offset calibration procedure performed on the SRM PowerMeter prior to each test, to determine V˙O2peak. Starting at 100 W, there was a stepwise increase in power of 20 W.min⁻¹. Expired gases were obtained using a breath by breath gas analyser (Metamax 3X, Cortex, Germany). HR and RPE were taken at the end of every stage. Anaerobic threshold was computer-determined with additional visual inspection to determine the first breakpoint in ventilatory parameters.

Twenty-four hours later, participants performed a TTE at an intensity corresponding to 80% peak power output (PPO) attained during the pre-SIT assessment of V˙O2peak, on a cycle ergometer at a target cadence of 80 revs.min⁻¹ (Monark, model 864, Sweden). The test was terminated at volitional exhaustion when the participants' cadence fell below 40 revs^.min⁻¹; exercise duration (seconds) was then determined. Capillary blood was collected from the fingertip pre and 2 min post TTE for analysis of blood lactate (2300 Stat Plus, YSI Life Sciences, USA).

Prior to each V˙O2peak test 10 mL of blood was collected from the ante-cubital fossa. Whole blood (~50 μL) was divided into two heparinised capillary tubes (Hawksley & Sons Ltd., England) then centrifuged (Hematospin 1300, Hawksley & Sons Ltd., England) at 1000 rpm for 1.5 min to calculate the haematocrit using a micro haematocrit reader (Hawksley & Sons Ltd., England); the average of duplicate samples was recorded. Hemoglobin concentration (B-Hemoglobin Photometer, Hemocue, Sweden) was determined via the average of triplicate samples (B-Hemoglobin Microvettes, Hemocue, Sweden). The remaining blood was transferred into two 5 mL EDTA tubes and centrifuged at 5000 rpm for 10 min. Plasma was then extracted into microvettes and stored at –86°C. IL-6 and TNFα concentrations were analyzed using Enzyme-linked immunosorbent assays in accordance with manufacturer instructions (DuoSet ELISA Development System; R&D Systems Inc., Abingdon, UK) with corrections made for the change in plasma volume (Dill and Costill, 1974). The technical error of measurement (TEM) between duplicate samples for IL-6 was 7.1%, with a unit error value of 2.76 pg.mL⁻¹ and for TNFα it was 4.1%, with a unit error value of 518.7 pg.mL⁻¹.

Each WAnT, performed 24 h following the TTE, consisted of 30 s of “all out” maximal cycling on a friction-loaded cycle ergometer (Monark Ergomedic Peak Bike 894e, Monark Exercise AB, Sweden). The load was calculated as 7.5% body mass [0.075 kg/kg.bm⁻¹, (Bar-Or, 1987)]. The onset of each sprint was marked with a “3-2-1. GO!” countdown, and participants were instructed to cycle for ~2 s against the ergometers inertial resistance before the full load was released at 70 rpm. Participants were required to stay seated on the saddle and were verbally encouraged throughout the test. Each sprint was followed by a 4 min recovery period—participants were required actively recover (unloaded cycling at 60 revs.min⁻¹). Peak power output, average 30-s power output (total work), and fatigue index [also known as rate of power decline; ((Peak Power Output–Min Power Output)/Peak Power Output) × 100) (%)] were recorded by Monark Anaerobic Test software (ver. 3.2.7.0, Monark Exercise AB, Sweden).

All SIT was performed on a cycle ergometer (Monark, model 864, Sweden) against a resistance of 0.075 kg.kg⁻¹ body mass, from a rolling start of 70 revs·min⁻¹. Participants were verbally encouraged throughout. The sprints were interspersed with a 4 min active recovery period of cycling at 60 W. Power measures were recorded using Monark Anaerobic Test software (Monark, Sweden) continuously throughout the sprints. The number of sprints increased from four to seven over the 2 weeks (total six sessions). Training days were interspersed with one rest day (Figure 1). SpO₂ and HR were monitored using a finger pulse oximeter (Nonin 2500, Nonin Medical Inc., USA) 1 min after every sprint.

Data were tested for normality, skewness and kurtosis. Data were normally distributed unless otherwise stated. A Two Way Mixed Design ANOVA was performed separately on each of the independent variables; V˙O2peak, TTE, Peak Power, Mean Power, Fatigue index (from WAnT test), IL-6, TNFα, Hb, and Hct, to determine whether there was a significant change between the three conditions (HYP, NORM, and CON) over two time-points (pre and post). The mean sessional recovery HR and SpO₂ observed following each SIT were analyzed using a mixed 2-way ANOVA using the Greenhouse-Geisser correction to determine whether there was a significant change between the two training conditions (HYP and NORM) over the six SIT sessions. Adjusted Bonferroni comparisons were used as post-hoc analyses for all ANOVA. Partial eta squared was used to calculate effect sizes (np²; small = 0.01, medium = 0.06, large = 0.13) were calculated to analyse the magnitude and trends with data. All data were reported as Mean ± SD. All statistical tests followed a significance level of p < 0.05. The statistical package used was SPSS (SPSS Inc., Chicago, USA, version 20.0).

V˙O2peak was significantly different from pre to post-training (p = 0.001, np² = 0.44), and between different training groups (p = 0.002, np² = 0.28, Figure 2). Post-hoc analysis observed increases in HYP (p = 0.001; +0.39 ± 0.29 L.min⁻¹; 43.6 ± 8.0 to 48.8 ± 9.2 mL.kg⁻¹.min⁻¹) and NORM (p = 0.002; +0.32 ± 0.38 L.min⁻¹; 42.2 ± 8.6 to 46.0 ± 7.5 mL.kg⁻¹.min⁻¹), but not for CONT (p = 0.906; 42.1 ± 9.7 to 42.2 ± 9.7 mL.kg⁻¹.min⁻¹). Relative power at V˙O2peak (W.kg⁻¹) was greater pre to post (p = 0.001, np² = 0.42) overall and for the pre-post*group interaction (p = 0.004, np² = 0.25). Post-hoc analysis observed increases in HYP (p = 0.001; 3.90 ± 0.72 to 4.20 ± 0.80 W.kg⁻¹) and NORM (p = 0.001; 3.54 ± 0.73 to 3.86 ± 0.86 W.kg⁻¹), but not for CONT (p = 0.872; 3.75 ± 0.56 to 3.76 ± 0.70 W.kg⁻¹).

The anaerobic threshold (AT) increased pre to post SIT (p = 0.001, np² = 0.30) overall and for the pre-post*group interaction (p = 0.001, np² = 0.29). Post-hoc analysis only observed increases in HYP (p = 0.001; 22.6 ± 4.1 to 24.6 ± 4.2 mL.kg⁻¹.min⁻¹) and not NORM (p = 0.050; 22.9 ± 4.7 to 24.0 ± 4.8 mL.kg⁻¹.min⁻¹) or CONT (p = 0.417; 22.7 ± 5.6 to 22.4 ± 4.9 mL.kg⁻¹.min⁻¹). Relative power at AT was greater pre to post-training (p = 0.001, np² = 0.313) overall and for the pre-post*group interaction (p = 0.006, np² = 0.23). Post-hoc analysis observed increases in both HYP (p = 0.001; 2.00 ± 0.36 to 2.26 ± 0.45 W.kg⁻¹) and NORM (p = 0.017; 1.88 ± 0.40 to 2.04 ± 0.49 W.kg⁻¹) but no change in CONT (p = 0.925; 1.98 ± 0.35 to 1.97 ± 0.37 W.kg⁻¹).

TTE was significantly different from pre to post test (p = 0.001, np² = 0.82) overall, and for the pre-post*group interaction (p = 0.001, np² = 0.64, Figure 2). Post-hoc analysis observed increases in HYP (p = 0.001; 633 ± 330 to 787 ± 326 s) and NORM (p = 0.001; 589 ± 373 to 729 ± 351 s), but not for CONT (p = 0.212; 607 ± 280 to 620 ± 274 s).

Peak power was not different during the WAnT from pre to post-training (p = 0.530, np² = 0.010) or between different training groups (CONT; 11.2 ± 3.0 to 10.9 ± 2.2 W.kg⁻¹, NORM; 11.0 ± 1.3 to 11.4 ± 1.4 W.kg⁻¹, HYP; 11.7 ± 2.4 to 11.9 ± 2.4 W.kg⁻¹) (p = 0.052, np² = 0.141).

Similarly, mean power was not different from pre to post-training (p = 0.653, np² = 0.005), or between training groups (CONT; 3.8 ± 0.8 to 3.7 ± 0.8 W.kg⁻¹, NORM; 4.0 ± 1.1 to 4.1 ± 0.9 W.kg⁻¹, HYP; 3.6 ± 1.3 to 3.8 ± 1.2 W.kg⁻¹) (p = 0.319, np² = 0.057).

Fatigue Index was found to be significantly reduced from pre to post-training (p = 0.001, np² = 0.968), although there was no significant difference between training groups CONT; 62.7 ± 10.7 to 62.9 ± 12.3%, NORM; 63.5 ± 9.2 to 61.8 ± 9.4%, HYP; 65.1 ± 12.6 to 62.3 ± 10.1%) (p = 0.851, np² = 0.008).

Hb was significantly different from pre to post-training (p = 0.036, np² = 0.11). However, this increase was not different between training groups (p = 0.082, np² = 0.12) for CONT (14.6 ± 1.5 to 14.6 ± 1.5 g.dL⁻¹), NORM (14.3 ± 1.5 to 14.3 ± 1.3 g.dL⁻¹), and HYP (14.7 ± 1.8 to 15.0 ± 1.7 g.dL⁻¹).

Hct was not different from pre to post-training (p = 0.701, np² = 0.00) or between groups (p = 0.215, np² = 0.08) for CONT (44.0 ± 2.8 to 43.7 ± 2.4%), NORM (44.9 ± 2.1 to 45.0 ± 2.7%), and HYP (45.7 ± 2.8 to 46.0 ± 3.1%).

Blood lactate increased in all TTE tests (p = 0.001, np² = 0.29). Post TTE blood lactate values increased significantly with NORM (p = 0.01 5.07 ± 0.77 to 5.62 ± 1.01 mmol.L⁻¹), and HYP training (p = 0.010; 5.24 ± 0.9 to 5.76 ± 0.82 mmol.L⁻¹), but not in CONT (p = 0.101; 6.42 ± 0.8 to 6.52 ± 0.95 mmol.L⁻¹).

IL-6 was significantly different from pre to post test (p = 0.001, np² = 0.39) and this increase was different between training groups (p = 0.007, np² = 0.23). Post-hoc analysis observed increases for HYP (p = 0.001; 1.7 ± 0.2 to 2.0 ± 0.2 pg.mL⁻¹) and NORM (p = 0.003; 1.7 ± 0.2 to 2.0 ± 0.3 pg.mL⁻¹), but not for CONT (p = 0.836; 1.7 ± 0.2 to 1.7 ± 0.2 pg.mL⁻¹).

TNFα was significantly different from pre to post test (p = 0.006, np² = 0.175), however, was not significantly different between training groups (p = 0.151, np² = 0.09) (NORM; 3.0 ± 0.6 to 3.4 ± 0.9 pg.mL⁻¹; HYP; 3.1 ± 0.8 to 3.3 ± 0.6 pg.mL⁻¹; CONT; 2.7 ± 0.7 to 2.8 ± 0.6 pg.mL⁻¹).

Recovery HR was not different between sessions (p = 0.250; np² = 0.11) or for the different training groups (p = 0.420; np² = 0.04, Figure 3). SpO₂ was different between sessions (p = 0.001; np² = 0.30) and significantly less with HYP training (p = 0.001; np² = 0.20). Post-hoc analysis of SpO₂ is presented in Figure 3 for clarity.

In the present study V˙O2peak, TTE, and power at the AT increased following SIT in HYP and NORM suggesting improved oxidative phosphorylation had occurred (Burgomaster et al., 2005, 2006). Interestingly, when considering AT expressed at mL.kg⁻¹.min⁻¹ this adaptation only occurred in HYP (+9.5%), not NORM (+5.0%). Puype et al. (2013) acknowledged that their 6-week intervention improved the power corresponding to the AT (quantified from a 4 mmol.L⁻¹ lactate concentration) by ~7% (in normoxia) and 9% (in hypoxia) only following hypoxic, and not normoxic training. This adaptation was associated with an increase in muscle phosphofructokinase (PFK; hypoxia = +59%, normoxia = +17%). It was reported that VO_2max increased (hypoxia = +7.4%; normoxia = +5.8%) and TTE improved (hypoxia = +5.0%; normoxia = +2.9%) compared to controls, but with no difference when performing the training in hypoxia vs. normoxia (Puype et al., 2013). These observations are in line with our experiment. Our data is also supportive of an increased AT following SIT (Puype et al., 2013), and therefore increased glycolytic capacity via elevated PFK. Methodological differences in identifying the threshold, and the three-fold protocol duration of Puype et al. (2013) provide a rationale for the contrast between significant changes in the power at the AT following training in hypoxia (Puype et al., 2013) and the absence of a further improvement in power at the AT in comparison to normoxia as observed by ourselves. The improved AT (mL.kg⁻¹.min⁻¹) following SIT in hypoxia in comparison to SIT in normoxia in our study may have also been acknowledged by Puype et al. (2013), unfortunately this was not quantified. SIT, or similar training, elicits known metabolic adaptations, e.g., increased oxidative (Gibala et al., 2006) and glycolytic enzyme activity (Talanian et al., 2007; Daussin et al., 2008), improved muscle buffering capacity, elevated intramuscular glycogen content (Burgomaster et al., 2005) and increased skeletal muscle capillarisation (Puype et al., 2013). These are likely induced from the performance of the sprints within the protocol, with hypoxia potentiating greater metabolic disturbances in vs. normoxia (Faiss et al., 2013b; Galvin et al., 2013; Puype et al., 2013). Even greater improvements in glycolytic function may have been identified by increased mean power and improved fatigue index during the WaNT (Puype et al., 2013), however this was not case suggesting the mode of training was not structured effectively to facilitate this. Increased lactate in response to improved TTE suggests a greater tolerance to metabolic acidosis via group III/IV afferents (Amann et al., 2015) with SIT not demonstrating improved lactate clearance as supported by evidence elsewhere (Juel et al., 2004). The absence of a difference in peak and mean power, and fatigue index during the WAnT demonstrate that SIT in normoxia and hypoxia was effective at improving aerobic metabolic pathways. Mechanisms supporting the physiological (V˙O2peak) and performance (TTE) responses in our normobaric hypoxic environment do not appear hematological given the lack of intragroup difference in Hb and Hct (Table 2), this is in agreement with the proposal of others (Richalet and Gore, 2008), reinforcing the metabolic adaptive pathway. The measurement of Hb and Hct would however be improved by assessing total Hb mass thus accounting for changes otherwise lost when measuring the concentration (Burge and Skinner, 1995; Schmidt and Prommer, 2005). Based upon the expected lack of hematological adaptations between groups, aforementioned modulators of oxygen utilization at the muscle are most likely improved by SIT irrespective of the FiO₂ in which it is performed. Interestingly, improvements in SpO₂ were observed within the HYP group by the 6th session (Figure 3). This suggests some level of acclimation to hypoxia had occurred. SIT may therefore be effective at mitigating desaturation known to occur during repeated/intermittent sprint performance in hypoxia (Bowtell et al., 2014; Turner et al., 2014). Accordingly, future work investigating training adaptations to SIT could consider the benefits of hypoxic SIT to prepare athletes, rather than untrained individuals as in the present experiment, for competition in hypoxia (Girard et al., 2013; Millet et al., 2013). Concurrent mechanistic work may also wish to consider whether our equal pre to post-intervention measures of Hb and Hct data confirms a lack of hematological adaptation i.e., HB_mass, in favor of improved metabolic pathways (Faiss et al., 2013b). Additionally exploration of the mechanisms by which preservation of SpO₂ occurs in response to SIT in hypoxia may also be considered. With no clear additional benefit of performing 2 weeks of SIT in hypoxia rather than normoxia (on V˙O2peak, and TTE performance lasting ~10 min, Table 2), implementing this training appears largely unreasoned from a performance perspective at the present time, particularly given the challenge of facilitating training in this environment.

The increase in basal IL-6 following both normoxic and hypoxic SIT (Table 2) reflects the intensity and duration of the activity as widely observed elsewhere (Fischer, 2006). The functional significance of an increased IL-6 is complex (Gleeson and Bishop, 2000), with increased exercise requirements (Croft et al., 2009) and increasing metabolic stress, e.g., via hypoxia in isolation, or hypoxia related increases in the relative exercise intensity (Schobersberger et al., 2000) typically eliciting larger responses. IL-6 has an important anti-inflammatory and adaptation signaling role during the post-exercise recovery phase (Svendsen et al., 2016), with a greater increase in IL-6 post-hypoxic exercise reflective of a greater training stress (Fischer, 2006; Scheller et al., 2011). A reduction of IL-6 is a known training adaptation (Fischer, 2006); the elevation of the cytokine 48 h following the final training session however indicates that recovery/adaptation was incomplete. Irrespective of the consequential effects of increased basal IL-6, the current data appeases concerns that training in hypoxia as having an impairment upon individuals when compared to equivalent normoxic training. The increase in TNF-α alongside IL-6 is similar to other data demonstrating relationships between these inflammatory biomarkers and exercise (Gleeson and Bishop, 2000). Interestingly, a similar magnitude of inflammatory response to SIT was observed for TNFα as IL-6, however this was not statistically different to controls despite the ~12% difference between NORM and HYP, and CON. This disparity between IL-6 and TNFα may be due to the greater within group variability observed with this inflammatory marker (~25%) or the lack of hypoxia specific TNFα induction during passive (Turner et al., 2016) or active (Svendsen et al., 2016) exposures. Nonetheless performing SIT in hypoxia did not exacerbate the inflammatory response in comparison to normoxia, and is therefore unlikely to be detrimental to subsequent training or performance. A more precise quantification of training load within each group, and subsequently ensuring equality of load between groups, would give further confidence in the equality of inflammatory responses to SIT performed in either normoxia or hypoxia. Should absolute or relative internal/external training load be different between SIT performed in normoxia or hypoxia, then this may influence the interpretation of the inflammatory markers and suggest that hypoxia reduces or exacerbates the responses.

Similar basal inflammatory markers IL-6 and TNFα 24 h post the final SIT suggests that the increased stress of training in hypoxia is equal to that of normoxia and would not be detrimental to the individual. This equality of cytokine response is in agreement with the comparable magnitude of increases in IL-6 (hypoxia = +57%, normoxia = +56%) and lack of change in TNFα 2 h after a 75 min submaximal cycle in either condition (Svendsen et al., 2016). Accordingly the benefits of hypoxic SIT to prepare athletes for competition in hypoxia (Millet et al., 2013) can be determined at least equal to that of equivalent training in normoxia, without further compromising subsequent activities. Given the abundance of cellular and molecular pathways associated with SIT and HIIT, and hypoxia, this experiment presents a constrained overview of the responses. To optimize the application of SIT in hypoxia, future work should consider measurement of a wider spectrum of blood and muscle markers of training adaptations associated with SIT, and hypoxia both in isolation, and in combination. Additionally, further analysis of the impact of SIT in hypoxia on stress markers should be considered at a basal level (as determined within the present experiment) but also regarding the kinetics of a within session increase and the subsequent time-course to return to baseline prior to, and beyond our 48 h measurement point.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RF and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.