Acute exercise activates the AHR in peripheral blood mononuclear cells in an intensity-dependent manner.

The kynurenine pathway (KP) of tryptophan degradation generates several metabolites such as kynurenine (KYN) or kynurenic acid (KA) that serve as endogenous ligands of the aryl hydrocarbon receptor (AHR). Due to its distinct biological roles particularly modulating the immune system, the AHR is a current therapeutic target across different inflammation-related diseases. Here, we show an acute exercise-induced increase in AHR ligand availability on a systemic level and a kynurenine pathway activation in peripheral blood mononuclear cells (PBMCs). Concurrently, the AHR is activated in PBMCs following acute exercise. Exercise effects on both, kynurenic acid and AHR activation in PBMCs were greater in response to high-intensity interval exercise (HIIE) (50 min, six 3-min intervals at 90% V̇o2peak, and 3-min intervals at 50% V̇o2peak in between) compared with workload-matched moderate-intensity continuous exercise (MICE) (50 min). In conclusion, these data indicate a novel mechanistic link in how exercise modulates the immune system through the kynurenine pathway-AHR axis, potentially underlying exercise-induced benefits in various chronic diseases.NEW & NOTEWORTHY The findings of this study show that acute endurance exercise activates a receptor that has been described to integrate metabolic signals into the immune system. We uncover a potential mechanistic link on how exercise modulates the immune system through the kynurenine pathway-AHR axis, potentially underlying exercise-induced benefits in various chronic diseases and of relevance for other cell types.

HIIT Induces Stronger Shifts within the Peripheral T Cell Compartment Independent of Sex.

Acute exercise induces changes within the T-cell compartment, especially in cytotoxic CD8+ memory subsets, depending on exercise intensity and duration. It is unclear whether exercise-induced changes in major T-cell subsets differ in response to acute high-intensity interval training (HIIT) or moderate-intensity continuous training (MICT) and whether sex-specific effects exist. Twenty-four recreationally active runners (females: n=12, 27.8±4.1years, 54.4±4.6 ml*kg-1*min-1; males: n=12, 31.6±3.8years, 58.9±7.7 ml*kg-1*min-1) participated in this randomized controlled crossover study, and conducted an energy- and duration-matched HIIT and MICT session. Blood was sampled before (T1), immediately (T2) and 1 h after exercise (T3). Flow cytometry was used to identify T-cell populations. HIIT decreased the proportion of CD8+ T-cells more pronounced at T3 compared to MICT (p=0.007), induced a significantly stronger increase in the CD8+ effector memory (TEM) cell proportion at T2 (p=0.032), and decreased CD4+ central memory proportion more pronounced at T2 (p=0.029). A decrease below baseline CD8+ TEM proportion at T3 was observed only after HIIT (p<0.001). No interaction effects between sexes were revealed. Taken together, HIIT represents a more potent stimulus to induce shifts mainly within the cytotoxic CD8+ T-cell compartment, thereby giving implications to investigate the role of HIIT on the cell´s effector phenotype and function in more detail.

Intensity- and time-matched acute interval and continuous endurance exercise similarly induce an anti-inflammatory environment in recreationally active runners: focus on PD-1 expression in Tregs and the IL-6/IL-10 axis.

PURPOSE: Acute exercise elicits a transient anti-inflammatory state during the early recovery period. Since recent studies reported on regimen-specific effects on immune-related humoral factors and cellular subsets, this study compared the effects of intensity- and time-matched acute interval and continuous exercise on peripheral anti-inflammatory cellular and humoral immune parameters with a particular focus on the PD-1 expression in CD4+ regulatory T cells (Tregs). METHODS: Twenty-four recreationally active runners (age: 29.7 ± 4.3 years, BMI: 22.2 ± 2.4, VO2peak: 56.6 ± 6.4 ml × kg-1 × min-1) participated in this crossover RCT. Each subject conducted a moderate continuous (MCE) and a high-intensity interval exercise (HIIE) session in a counterbalanced design. Blood was drawn before, immediately after, and 1 h after exercise. Treg subsets and levels of PD-1 and Foxp3 were assessed by flow cytometry. Serum levels of IL-10 and IL-6 were quantified by ELISA. RESULTS: PD-1 levels on Tregs increased within the recovery period after HIIE (p < .001) and MCE (p <  0.001). Total counts of Tregs (HIIE: p = 0.044; MCE: p = .021), naïve Tregs (HIIE: p  < 0.001; MCE: p  < 0.001), and PD-1+ effector Tregs (eTregs) (HIIE: p = .002) decreased 1 h after exercise. IL-10 increased 1 h after HIIE (p < 0.001) and MCE (p = 0.018), while IL-6 increased immediately after both HIIE (p = 0.031) and MCE (p = 0.021). Correlations between changes in IL-6 and IL-10 (p = 0.017, r = 0.379) and baseline VO2peak and Treg frequency (p = 0.002, r = 0.660) were identified. CONCLUSION: This is the first study that investigates PD-1 expression in circulating Tregs after acute exercise, revealing an increase in PD-1 levels on eTregs during the early recovery period after intensity- and time-matched HIIE and MCE. Future studies are needed to investigate the PD-1 signalosome in eTregs, together with the expression of key effector molecules (i.e., IL-10, TGF-ß, IL-35, CTLA-4) to elucidate PD-1-dependent changes in cellular function. Based on changes in serum cytokines, this study further reveals a regimen-independent establishment of an anti-inflammatory milieu and underpins the role of the IL-6/IL-10 axis.

Maternal knowledge of recommendations for safe infant sleep and intentions for implementation - a cross sectional analysis of data from the KUNO-Kids birth cohort study.

OBJECTIVES: Despite major advances in prevention, sudden infant death syndrome (SIDS) remains an important cause of infant mortality. The aim of our study was to determine actual knowledge and intentions to implement SIDS prevention measures among new mothers and to identify potential knowledge gaps for improved postpartum counselling strategies. METHODS: Data was collected in a standardized interview from participants of the KUNO-Kids birth cohort study before discharge from maternity ward. The mothers did not receive any specific teaching prior to the interview. RESULTS: The majority of 2,526 interviewed mothers were able to actively report important recommendations for safe infant sleep, including the exclusive face-up position. However, 154 mothers (9%) intended to position the newborn face-down sometimes or often. The most frequently envisaged sleeping furniture was a bedside sleeper (n=1,144, 47%), but 2.2% of mothers indicated that the intended default sleeping place for the newborn would be the parents' bed (which is discouraged by the recommendations). For 43% of the infants (n=1,079), mothers planned to have loose objects in the bed and 189 mothers (7%) intended to use a loose blanket. 22% of infants (n=554) will live in a household with a smoker. Multivariate regression showed a significant association of "good knowledge" with maternal age and with not being a single parent, whereas the household size was negatively associated. CONCLUSION: Although the majority of mothers in our birth cohort were aware of many recommendations for safe infant sleep, our data also uncovered weaknesses in SIDS prevention knowledge and point to specific areas with potential for improved counselling.

Cellular immune response to acute exercise: Comparison of endurance and resistance exercise.

OBJECTIVES: Exercise-induced cellular mobilization might play a role in treatment and prevention of several diseases. However, little is known about the impact of different exercise modalities on immune cell mobilization and clinical cellular inflammation markers. Therefore, the present study aimed to investigate differences between acute endurance exercise (EE) and resistance exercise (RE) on cellular immune alterations. METHODS: Twenty-four healthy men conducted an acute EE (cycling at 60% of peak power output) and RE (five exercise machines at 70% of the one-repetition maximum) session lasting 50 minutes in randomized order. Blood samples were collected before, after and one hour after exercise cessation. Outcomes included counts and proportions of leukocytes, neutrophils (NEUT), lymphocytes (LYM), LYM subsets, CD4/CD8 ratio, and the clinical cellular inflammation markers NEUT/LYM ratio (NLR), platelets/LYM ratio (PLR), and systemic immune inflammation index (SII). RESULTS: Alterations in all outcomes were revealed except for CD8+ T cells, CD4/CD8 ratio, NLR, and PLR. EE induced a stronger cellular immune response and provoked alterations in more immune cell populations than RE. SII was altered only after EE. CONCLUSION: An acute EE session causes a stronger mobilization of immune cells than RE. Additionally, SII represents an integrative marker to depict immunological alterations.

Exercise and the Kynurenine pathway: Current state of knowledge and results from a randomized cross-over study comparing acute effects of endurance and resistance training.

INTRODUCTION: The essential amino acid tryptophan (TRP) is primarily degraded through the kynurenine (KYN) pathway, which is dysregulated in several chronic diseases. KYN pathway metabolites have immune- and neuro-modulatory properties and are involved in th de novo synthesis of nicotinamide adenine dinucleotide (NAD+). Currently, little evidence exists demonstrating that physical exercise may influence this pathway. However, differences between acute and chronic stimuli as well as the influence of exercise modalities remain to be investigated. Here, we provide an overview of existing studies and present results of a randomized cross-over trial on acute effects of a single-bout of resistance and endurance exercise. METHODS: 24 healthy male adults conducted both an acute endurance exercise (EE) and resistance exercise (RE) session. Blood samples were collected before, immediately after and one hour after cessation of each exercise session. Outcomes comprised serum levels of TRP, KYN, kynurenic acid (KA), quinolinic acid (QA) and calculated ratios. Gene expression of the enzymes indoleamine 2,3 dioxygenase (IDO) 1 and kynurenine aminotransferase (KAT) 4 was measured in peripheral blood mononuclear cells (PBMCs). Moreover, serum concentrations of the potential KYN pathway mediators interleukin (IL)-6 and cortisol were determined. Finally, we investigated baseline correlations between immune cell subsets, potential mediators and initial KYN pathway activation outcomes. RESULTS: The KYN/TRP ratio correlated positively with IL-6 and CD56bright NK-cells and negatively with CD56dim NKcells. Expression of IDO1 in PBMCs correlated positively with IL-6, regulatory T-cells and CD56bright NK-cells, whereas negative correlations to cytotoxic T-cells and CD56dim NKcells were revealed. A significant time effect on KYN/TRP ratio was detected for RE. Regarding KA and KA/KYN ratio, an increase after exercise followed by a decrease at the follow- up measurement was revealed in EE. KAT4 expression also increased after exercise in EE. Moreover, elevated QA levels were observed after the EE session. CONCLUSIONS: In contrast to chronic exercise interventions, single-bouts of endurance exercise provoke acute alterations on KYN pathway outcomes in humans. Our results indicate that EE induces stronger alterations than RE. Enhanced conversion of KYN to both, KA and QA suggest a peripheral KYN clearance, thereby preventing pathological accumulation within the CNS. Future acute and chronic exercise studies are needed to examine the role of NAD+ synthesis starting with TRP and the interplay between KYN pathway activation and mid- to long-term immunological modulations.

Influence of combined functional resistance and endurance exercise over 12 weeks on matrix metalloproteinase-2 serum concentration in persons with relapsing-remitting multiple sclerosis - a community-based randomized controlled trial.

BACKGROUND: The relevance of regular moderate to intense exercise for ameliorating psychomotor symptoms in persons with multiple sclerosis (pwMS) is becoming increasingly evident. Over the last two decades, emerging evidence from clinical studies and animal models indicate immune regulatory mechanisms in both periphery and the central nervous system that may underlie these beneficial effects. The integrity of the blood-brain barrier as the main structural interface between periphery and brain seems to play an important role in MS. Reducing the secretion of proteolytic matrix metalloproteinases (MMP), i.e. MMP-2, as disruptors of blood-brain barrier integrity could have profound implications for MS. METHODS: In this two-armed randomized controlled trial 64 participants with relapsing-remitting MS (RRMS) (EDSS 0-4.0) will be allocated to either an intervention group or a passive wait list control group. The intervention group will perform 60 min of combined functional resistance and endurance exercises 3x per week over a period of 12 weeks in a community-based and publicly available setting. Changes in serum concentration of MMP-2 will be the primary outcome. Secondary outcomes are numbers of immune cell subsets, soluble (anti-) inflammatory factors, physical capacity, cognitive performance, physical activity behavior, gait performance, and patient-reported outcomes. All outcome measures will be assessed at baseline and after week 12 with an additional blood sampling before, during and immediately after a single training session in week 6. DISCUSSION: To our knowledge, this will be the first RCT to investigate both the acute and chronic effects of a community-based intense functional resistance and endurance exercise regimen in persons with RRMS. Combining analysis of biological and cognitive or psychological outcomes may provide a better understanding of the MS-specific symptomology. TRIAL REGISTRATION: DRKS00017091; 05th of April, 2019; International Clinical Trials Registry Platform.

Comprehensive Analytics of Actovegin® and Its Effect on Muscle Cells.

The ingredients of Actovegin® were analyzed and its effects on the muscle cell proliferation were investigated. C2C12 myoblasts were cultured in medium. Actovegin® was added in five different concentrations (1, 5, 25, 125, and 250 µg) to the differentiation medium. The formations of proliferation factor Ki67 and myosin heavy chains were measured by immunofluorescence. The first primary antibody was anti-Ki67 and anti-Mf20. Cells were washed and treated with the second fluorochrome. Thirty-one Actovegin® ingredients were found to contain significantly higher concentrations and twenty-nine ingredients were found to contain significantly lower concentrations, compared to the mean ranges as described in the literature for the normal physiological concentrations in human adult serum/plasma. A significant increase in the formation of Ki67 was observed in Actovegin® groups, compared to controls. The mean area of myotubes was significantly increased in Actovegin® groups. A significant decrease in the number of myotubes was observed. An increased myotube size (fusion) was observed. The intensity of Mf20 was significantly increased in Actovegin® groups. It could be demonstrated that Actovegin® contains many physiological substances in significantly higher and some in lower concentrations compared to human adult serum. Furthermore, it could be shown that Actovegin® improves muscle cell proliferation.

Conventional testing methods produce submaximal values of maximum oxygen consumption.

BACKGROUND: This study used a novel protocol to test the hypothesis that a plateau in oxygen consumption (VO(2 max)) during incremental exercise testing to exhaustion represents the maximal capacity of the cardiovascular system to transport oxygen. METHODS: Twenty-six subjects were randomly divided into two groups matched by their initial VO(2 max). On separate days, the reverse group performed (i) an incremental uphill running test on a treadmill (INC(1)) plus verification test (VER) at a constant workload 1 km h(-1) higher than the last completed stage in INC(1); (ii) a decremental test (DEC) in which speed started as same as the VER but was reduced progressively and (iii) a final incremental test (INC(F)). The control group performed only INC on the same days that the reverse group was tested. RESULTS: VO(2 max) remained within 0.6 ml kg(-1) min(-1) across the three trials for the control group (p=0.93) but was 4.4% higher during DEC compared with INC(1) (63.9 ± 3.8 vs 61.2 ± 4.8 ml kg(-1) min(-1), respectively, p=0.004) in the reverse group, even though speed at VO(2 max) was lower (14.3 ± 1.1 vs 16.2 ± 0.7 km h(-1) for DEC and INC(1), respectively, p=0.0001). VO(2 max) remained significantly higher during INC(F) (63.6 ± 3.68 ml kg(-1) min(-1), p=0.01), despite an unchanged exercise time between INC(1) and INC(F). CONCLUSION: These findings go against the concept that a plateau in oxygen consumption measured during the classically described INC and VER represents a systemic limitation to oxygen use. The reasons for a higher VO(2) during INC(F) following the DEC test are unclear.

Comparison of Physiological Responses and Muscle Activity During Incremental and Decremental Cycling Exercise.

OBJECTIVE: To investigate whether a cycling test based on decremental loads (DEC) could elicit higher maximal oxygen uptake (V˙O2max) values compared with an incremental test (INC). DESIGN: Nineteen well-trained individuals performed an INC and a DEC test on a single day, in randomized order. METHODS: During INC, the load was increased by 20 W·min-1 until task failure. During DEC, the load started at 20 W higher than the peak load achieved during INC (familiarization trial) and was progressively decreased. Gas exchange and electromyography (EMG) activity (n = 11) from 4 lower-limb muscles were monitored throughout the tests. Physiological and EMG data measured at V˙O2max were compared between the 2 protocols using paired t tests. RESULTS: V˙O2max during the DEC was 3.0% (5.9%) higher than during INC (range 94%-116%; P = .01), in spite of a lower power output (-21 [20] W, P < .001) at V˙O2max. Pulmonary ventilation (P = .036) and breathing rate (P = .023) were also higher during DEC. EMG activity measured at V˙O2max was not different between tests, despite the lower output during DEC. CONCLUSIONS: A DEC exercise test produces higher V˙O2max in cycling compared with an INC test, which was accompanied by higher pulmonary ventilation and similar EMG activity. The additional O2 uptake during DEC might be related to extra work performed either by the respiratory muscles and/or the less oxidatively efficient leg muscles.

Physical Exercise as Kynurenine Pathway Modulator in Chronic Diseases: Implications for Immune and Energy Homeostasis.

Emerging evidence highlights the substantial role of the kynurenine pathway in various physiological systems and pathological conditions. Physical exercise has been shown to impact the kynurenine pathway in response to both single (acute) and multiple (chronic) exercise training stimuli. In this perspective article, we briefly outline the current knowledge concerning exercise-induced modulations of the kynurenine pathway and discuss underlying mechanisms. Furthermore, we expose the potential involvement of exercise-induced kynurenine pathway modulations on energy homeostasis (eg, through de novo synthesis of NAD+) and finally suggest how these modulations may contribute to exercise-induced benefits in the prevention and treatment of chronic diseases.

Acute and chronic effects of exercise on the kynurenine pathway in humans - A brief review and future perspectives.

Tryptophan (TRP) is an essential amino acid. Metabolites of TRP have been identified as important mediators in immune regulation and function of the central nervous system. Inflammation strongly stimulates to the breakdown of TRP into Kynurenine (KYN), representing the initial step of the KYN pathway. Recently, exercise interventions have been able to demonstrate a modification of the KYN pathway plausibly by altering inflammation. However, modifications differ between acute and chronic exercise interventions. As such, this review examines the current studies that have investigated the effect of an acute (single bout) or chronic (training) exercise intervention on levels of TRP and KYN in both healthy and diseased populations.

Within-Season Distribution of External Training and Racing Workload in Professional Male Road Cyclists.

PURPOSE: To describe the within-season external workloads of professional male road cyclists for optimal training prescription. METHODS: Training and racing of 4 international competitive professional male cyclists (age 24 ± 2 y, body mass 77.6 ± 1.5 kg) were monitored for 12 mo before the world team-time-trial championships. Three within-season phases leading up to the team-time-trial world championships on September 20, 2015, were defined as phase 1 (Oct-Jan), phase 2 (Feb-May), and phase 3 (June-Sept). Distance and time were compared between training and racing days and over each of the various phases. Times spent in absolute (<100, 100-300, 400-500, >500 W) and relative (0-1.9, 2.0-4.9, 5.0-7.9, >8 W/kg) power zones were also compared for the whole season and between phases 1-3. RESULTS: Total distance (3859 ± 959 vs 10911 ± 620 km) and time (240.5 ± 37.5 vs 337.5 ± 26 h) were lower (P < .01) in phase 1 than phase 2. Total distance decreased (P < .01) from phase 2 to phase 3 (10911 ± 620 vs 8411 ± 1399 km, respectively). Mean absolute (236 ± 12.1 vs 197 ± 3 W) and relative (3.1 ± 0 vs 2.5 ± 0 W/kg) power output were higher (P < .05) during racing than training, respectively. CONCLUSION: Volume and intensity differed between training and racing over each of 3 distinct within-season phases.

The Hsp72 and Hsp90α mRNA Responses to Hot Downhill Running Are Reduced Following a Prior Bout of Hot Downhill Running, and Occur Concurrently within Leukocytes and the Vastus Lateralis.

The leukocyte heat shock response (HSR) is used to determine individual's thermotolerance. The HSR and thermotolerance are enhanced following interventions such as preconditioning and/or acclimation/acclimatization. However, it is unclear whether the leukocyte HSR is an appropriate surrogate for the HSR in other tissues implicated within the pathophysiology of exertional heat illnesses (e.g., skeletal muscle), and whether an acute preconditioning strategy (e.g., downhill running) can improve subsequent thermotolerance. Physically active, non-heat acclimated participants were split into two groups to investigate the benefits of hot downhill running as preconditioning strategy. A hot preconditioning group (HPC; n = 6) completed two trials (HPC1HOTDOWN and HPC2HOTDOWN) of 30 min running at lactate threshold (LT) on -10% gradient in 30°C and 50% relative humidity (RH) separated by 7 d. A temperate preconditioning group (TPC; n = 5) completed 30 min running at LT on a -1% gradient in 20°C and 50% (TPC1TEMPFLAT) and 7 d later completed 30 min running at LT on -10% gradient in 30°C and 50% RH (TPC2HOTDOWN). Venous blood samples and muscle biopsies (vastus lateralis; VL) were obtained before, immediately after, 3, 24, and 48 h after each trial. Leukocyte and VL Hsp72, Hsp90α, and Grp78 mRNA relative expression was determined via RT-QPCR. Attenuated leukocyte and VL Hsp72 (2.8 to 1.8 fold and 5.9 to 2.4 fold; p < 0.05) and Hsp90α mRNA (2.9 to 2.4 fold and 5.2 to 2.4 fold; p < 0.05) responses accompanied reductions (p < 0.05) in physiological strain [exercising rectal temperature (-0.3°C) and perceived muscle soreness (~ -14%)] during HPC2HOTDOWN compared to HPC1HOTDOWN (i.e., a preconditioning effect). Both VL and leukocyte Hsp72 and Hsp90α mRNA increased (p < 0.05) simultaneously following downhill runs and demonstrated a strong relationship (p < 0.01) of similar magnitudes with one another. Hot downhill running is an effective preconditioning strategy which ameliorates physiological strain, soreness and Hsp72 and Hsp90α mRNA responses to a subsequent bout. Leukocyte and VL analyses are appropriate tissues to infer the extent to which the HSR has been augmented.

Downhill running and exercise in hot environments increase leukocyte Hsp72 (HSPA1A) and Hsp90α (HSPC1) gene transcripts.

Stressors within humans and other species activate Hsp72 and Hsp90α mRNA transcription, although it is unclear which environmental temperature or treadmill gradient induces the largest increase. To determine the optimal stressor for priming the Hsp system, physically active but not heat-acclimated participants (19.8 ± 1.9 and 20.9 ± 3.6 yr) exercised at lactate threshold in either temperate (20°C, 50% relative humidity; RH) or hot (30°C, 50% RH) environmental conditions. Within each condition, participants completed a flat running (temperate flat or hot flat) and a downhill running (temperate downhill or hot downhill) experimental trial in a randomized counterbalanced order separated by at least 7 days. Venous blood samples were taken immediately before (basal), immediately after exercise, and 3 and 24 h postexercise. RNA was extracted from leukocytes and RT-quantitative PCR conducted to determine Hsp72 and Hsp90α mRNA relative expression. Leukocyte Hsp72 mRNA was increased immediately after exercise following downhill running (1.9 ± 0.9-fold) compared with flat running (1.3 ± 0.4-fold; P = 0.001) and in hot (1.9 ± 0.6-fold) compared with temperate conditions (1.1 ± 0.5-fold; P = 0.003). Leukocyte Hsp90α mRNA increased immediately after exercise following downhill running (1.4 ± 0.8-fold) compared with flat running (0.9 ± 0.6-fold; P = 0.002) and in hot (1.6 ± 1.0-fold) compared with temperate conditions (0.9 ± 0.6-fold; P = 0.003). Downhill running and exercise in hot conditions induced the largest stimuli for leukocyte Hsp72 and Hsp90α mRNA increases.

The efficacy of the self-paced V̇O2max test to measure maximal oxygen uptake in treadmill running.

The novel self-paced, cycle-based maximal oxygen uptake (V̇O2max) test (SPV) has been shown to produce higher V̇O2max values than standard graded exercise test (GXT) protocols. This study sought to ascertain whether these observations would also be apparent in a self-paced, treadmill-based test design. Fourteen trained male runners performed a standard GXT on a motorised treadmill and a self-paced V̇O2max test on a nonmotorised treadmill in a counter-balanced design. The GXT included a plateau verification and was designed to last between 8 and 12 min. The self-paced test included 5 × 2 min stages and allowed participants to set their own running speed based on fixed increments in rating of perceived exertion. Significantly higher V̇O2max values (t[13] = 3.71, p = 0.003) were achieved in the self-paced test (64.4 ± 7.3 mL · kg(-1) · min(-1)) compared with the GXT (61.3 ± 7.3 mL · kg(-1) · min(-1)), and 13 of the 14 participants achieved the same or higher V̇O2max values in the self-paced test. Higher (p = 0.01) maximum heart rates were observed in the GXT (191 ± 10 beats · min(-1) vs. 187 ± 7 beats · min(-1)), but no differences were observed in any other recorded variables. The self-paced V̇O2max test may provide a more valid means of measuring V̇O2max than the GXT and suggests that a V̇O2 plateau during a GXT does not always signify achievement of a definitive V̇O2max. These results provide further support that self-paced V̇O2max testing produces higher values for maximal oxygen uptake.