Introduction: Energetic demand from high-intensity exercise can easily exceed the ATP synthesis rates of mitochondria leading to a reliance on anaerobic metabolism. The reliance on anaerobic metabolism disrupts metabolic homeostasis via the accumulation of intracellular metabolites, most notably inorganic phosphate (Pi) and hydrogen ions (H + ). In the first few seconds of high-intensity exercise, intracellular pH is typically ~7.1 and free Pi ~10 mM; however, during severe fatigue pH can fall to ~6.2 and Pi can exceed 30 mM. These metabolites are well known to contribute to fatigue by inhibiting cross-bridge kinetics and altering Ca 2 + handling. Yet, despite their clear effects on contractile processes, it remains unknown whether Pi and H + also impair muscle performance by disrupting mitochondrial respiratory function in human skeletal muscle. Methods: Nine young adults (29 ± 5 yr; 6 females) completed two laboratory visits. During Visit 1, participants performed an incremental cycling test to exhaustion to determine peak oxygen consumption (VO 2 peak). On Visit 2, a vastus lateralis biopsy was obtained and a portion (~30 mg) was allocated for high-resolution respirometry (HRR). The sample was dissected into three longitudinal fiber bundles, to perform experiments in triplicate on each subject. Each bundle was further subdivided into four pieces (~1.5 mg) to ensure the same fibers were assessed in all four metabolite conditions: (a) pH 7.1, 10 mM Pi; (b) pH 7.1, 30 mM Pi; (c) pH 6.2, 10 mM Pi; and (d) pH 6.2, 30 mM Pi. O 2 flux was measured for Complex I (CI) leak respiration, CI oxidative phosphorylation (OXPHOS), CI+Complex II (CI+CII) OXPHOS, and CI+CII maximal electron transport system (ETS) capacity. Results: VO 2 peak was 39±6ml·kg - ¹·min - ¹. CI leak respiration did not differ between conditions (P=0.06). In contrast, there was a main effect of metabolite accumulation on O 2 flux in CI OXPHOS, CI+CII OXPHOS, and CI+CII ETS (all P< 0.002), whereby acidosis (pH 6.2) inhibited respiration, but Pi had no effect. Specifically, O 2 flux in CI OXPHOS was lower in pH 6.2, 10 mM Pi (−32%, P=0.017) and pH 6.2, 30 mM Pi (−29%, P=0.004) compared with pH 7.1, 10 mM Pi. O 2 flux in CI+CII OXPHOS was lower in pH 6.2, 30 mM Pi (−16%, P=0.037) compared to pH 7.1, 10 mM Pi. O 2 flux in CI+CII ETS was lower in pH 6.2, 10 mM Pi (−24%, P< 0.001) and pH 6.2, 30 mM Pi (−28%, P=0.005) compared to pH 7.1, 10 mM Pi. Conclusion: These data suggest that acidosis, but not inorganic phosphate, impairs mitochondrial O 2 flux during both oxidative phosphorylation and maximal electron transport system capacity in human skeletal muscle. The multifaceted and distinct ways in which H + and Pi disrupt muscle function at multiple levels highlight these metabolites as potential molecular targets for enhancing muscle performance and mitigating fatigue during high-intensity exercise. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Colosio et al. (Fri,) studied this question.