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When high-intensity exercise is performed until exhaustion a “functional reserve” (FR) or capacity to produce power at the same level or higher than reached at exhaustion exists at task failure, which could be related to reactive oxygen and nitrogen species (RONS)-sensing and counteracting mechanisms. Nonetheless, the magnitude of this FR remains unknown. Repeated bouts of supramaximal exercise at 120% of VO2max interspaced with 20s recovery periods with full ischaemia were used to determine the maximal FR. Then, we determined which muscle phenotypic features could account for the variability in functional reserve in humans. Exercise performance, cardiorespiratory variables, oxygen deficit, and brain and muscle oxygenation (near-infrared spectroscopy) were measured, and resting muscle biopsies were obtained from 43 young healthy adults (30 males). Males and females had similar aerobic (VO2max per kg of lower extremities lean mass (LLM): 166.7 ± 17.1 and 166.1 ± 15.6 ml kg LLM−1.min−1, P = 0.84) and anaerobic fitness (similar performance in the Wingate test and maximal accumulated oxygen deficit when normalized to LLM). The maximal FR was similar in males and females when normalized to LLM (1.84 ± 0.50 and 2.05 ± 0.59 kJ kg LLM−1, in males and females, respectively, P = 0.218). This FR depends on an obligatory component relying on a reserve in glycolytic capacity and a putative component generated by oxidative phosphorylation. The aerobic component depends on brain oxygenation and phenotypic features of the skeletal muscles implicated in calcium handling (SERCA1 and 2 protein expression), oxygen transport and diffusion (myoglobin) and redox regulation (Keap1). The glycolytic component can be predicted by the protein expression levels of pSer40-Nrf2, the maximal accumulated oxygen deficit and the protein expression levels of SOD1. Thus, an increased capacity to modulate the expression of antioxidant proteins involved in RONS handling and calcium homeostasis may be critical for performance during high-intensity exercise in humans.

The NF-κB signalling pathway plays a critical role in inflammation, immunity, cell proliferation, apoptosis, and muscle metabolism. NF-κB is activated by extracellular signals and intracellular changes in Ca2+, Pi, H+, metabolites and reactive oxygen and nitrogen species (RONS). However, it remains unknown how NF-κB signalling is activated during exercise and how metabolite accumulation and PO2 influence this process. Eleven active men performed incremental exercise to exhaustion (IE) in normoxia and hypoxia (PIO2:73 mmHg). Immediately after IE, the circulation of one leg was instantaneously occluded (300 mmHg). Muscle biopsies from m. vastus lateralis were taken before (Pre), and 10s (Post, occluded leg) and 60s after exercise from the occluded (Oc1m) and free circulation (FC1m) legs simultaneously together with femoral vein blood samples. NF-κB signalling was activated by exercise to exhaustion, with similar responses in normoxia and acute hypoxia, as reflected by the increase of p105, p50, IKKα, IκBβ and glutathione reductase (GR) protein levels, and the activation of the main kinases implicated, particularly IKKα and CaMKII δD, while IKKβ remained unchanged. Postexercise ischaemia maintained and stimulated further NF-κB signalling by impeding muscle reoxygenation. These changes were quickly reverted at the end of exercise when the muscles recovered with open circulation. Finally, we have shown that Thioredoxin 1 (Trx1) protein expression was reduced immediately after IE and after 1 min of occlusion while the protein expression levels of glutathione peroxidase 1 (Gpx1) and thioredoxin reductase 1 (TrxR1) remained unchanged. These novel data demonstrate that exercising to exhaustion activates NF-κB signalling in human skeletal muscle and regulates the expression levels of antioxidant enzymes in human skeletal muscle. The fast regulation of NF-κB at exercise cessation has implications for the interpretation of published studies and the design of new experiments.

Zynamite PX®, a mango leaf extract combined with quercetin, enhances exercise performance by unknown molecular mechanisms. Twenty-five volunteers were assigned to a control (17 males) or supplementation group (8 males, receiving 140 mg of Zynamite® + 140 mg quercetin/8 h for 2 days). Then, they performed incremental exercise to exhaustion (IE) followed by occlusion of the circulation in one leg for 60 s. Afterwards, the cuff was released, and a 30 s sprint was performed, followed by 90 s circulatory occlusion (same leg). Vastus lateralis muscle biopsies were obtained at baseline, 20 s after IE (occluded leg) and 10 s after Wingate (occluded leg), and bilaterally at 90 s and 30 min post exercise. Compared to the controls, the Zynamite PX® group showed increased basal protein expression of Thr287-CaMKIIδD (2-fold, p = 0.007) and Ser9-GSK3β (1.3-fold, p = 0.005) and a non-significant increase of total NRF2 (1.7-fold, p = 0.099) and Ser40-NRF2 (1.2-fold, p = 0.061). In the controls, there was upregulation with exercise and recovery of total NRF2, catalase, glutathione reductase, and Thr287-CaMKIIδD (1.2–2.9-fold, all p < 0.05), which was not observed in the Zynamite PX® group. In conclusion, Zynamite PX® elicits muscle signaling changes in resting skeletal muscle resembling those described for exercise training and partly abrogates the stress kinases responses to exercise as observed in trained muscles.

The Nrf2 transcription factor is induced by reactive oxygen and nitrogen species and is necessary for the adaptive response to exercise in mice. It remains unknown whether Nrf2 signalling is activated by exercise in human skeletal muscle. Here we show that Nrf2 signalling is activated by exercise to exhaustion with similar responses in normoxia (PIO2: 143 mmHg) and severe acute hypoxia (PIO2: 73 mmHg). CaMKII and AMPKα phosphorylation were similarly induced in both conditions. Enhanced Nrf2 signalling was achieved by raising Nrf2 total protein and Ser40 Nrf2 phosphorylation, accompanied by a reduction of Keap1. Keap1 protein degradation is facilitated by the phosphorylation of p62/SQSTM1 at Ser349 by AMPK, which targets Keap1 for autophagic degradation. Consequently, the Nrf2-to-Keap1 ratio was markedly elevated and closely associated with a 2-3-fold increase in Catalase protein. No relationship was observed between Nrf2 signalling and SOD1 and SOD2 protein levels. Application of ischaemia immediately at the end of exercise maintained these changes, which were reverted within 1 min of recovery with free circulation. While SOD2 did not change significantly during either exercise or ischaemia, SOD1 protein expression was marginally downregulated and upregulated during exercise in normoxia and hypoxia, respectively. We conclude that Nrf2/Keap1/Catalase pathway is rapidly regulated during exercise and recovery in human skeletal muscle. Catalase emerges as an essential antioxidant enzyme acutely upregulated during exercise and ischaemia. Post-exercise ischaemia maintains Nrf2 signalling at the level reached at exhaustion and can be used to avoid early post-exercise recovery, which is O2-dependent.

Prolonged or unusual exercise may cause exercise-induced muscle damage (EIMD). To test whether Zynamite®, a mango leaf extract rich in the natural polyphenol mangiferin, administered in combination with quercetin facilitates recovery after EIMD, 24 women and 33 men were randomly assigned to two treatment groups matched by sex and 5 km running performance, and ran a 10 km race followed by 100 drop jumps to elicit EIMD. One hour before the competition, and every 8 h thereafter for 24 h, they ingested placebo (728 mg of maltodextrin) or 140 mg of Zynamite® combined with 140 mg of quercetin (double-blind). Although competition times were similar, polyphenol supplementation attenuated the muscle pain felt after the competition (6.8 ± 1.5 and 5.7 ± 2.2 a.u., p = 0.035) and the loss of jumping performance (9.4 ± 11.5 and 3.9 ± 5.2%, p = 0.036; p = 0.034) and mechanical impulse (p = 0.038) 24 h later. The polyphenols attenuated the increase of serum myoglobin and alanine aminotransferase in men, but not in women (interaction p < 0.05). In conclusion, a single dose of 140 mg Zynamite® combined with 140 mg of quercetin, administered one hour before competition, followed by three additional doses every eight hours, attenuates muscle pain and damage, and accelerates the recovery of muscle performance.

The mango leaf extract rich in mangiferin Zynamite® improves exercise performance when combined with luteolin or quercetin ingested at least 48 h prior to exercise. To determine whether a single dose of Zynamite® administered 1 h before exercise increases repeated-sprint performance, 20 men and 20 women who were physically active were randomly assigned to three treatments following a double-blind cross-over counterbalanced design. Treatment A, 140 mg of Zynamite®, 140 mg of quercetin, 147.7 mg of maltodextrin, and 420 mg of sunflower lecithin; Treatment B, 140 mg of Zynamite®, 140 mg of quercetin, and 2126 mg of maltodextrin and Treatment C, 2548 mg of maltodextrin (placebo). Subjects performed three Wingate tests interspaced by 4 min and a final 15 s sprint after ischemia. Treatments A and B improved peak power output during the first three Wingates by 2.8% and 3.8%, respectively (treatment x sprint interaction, p = 0.01). Vastus Lateralis oxygenation (NIRS) was reduced, indicating higher O2 extraction (treatment × sprint interaction, p = 0.01). Improved O2 extraction was observed in the sprints after ischemia (p = 0.008; placebo vs. mean of treatments A and B). Blood lactate concentration was 5.9% lower after the ingestion of Zynamite® with quercetin in men (treatment by sex interaction, p = 0.049). There was a higher Vastus Lateralis O2 extraction during 60 s ischemia with polyphenols (treatment effect, p = 0.03), due to the greater muscle VO2 in men (p = 0.001). In conclusion, a single dose of Zynamite® combined with quercetin one hour before exercise improves repeated-sprint performance and muscle O2 extraction and mitochondrial O2. consumption during ischemia. No advantage was obtained from the addition of phospholipids.