This study tested the hypothesis that acidic pH inhibits oxidative ATP supply during exercise in hand (first dorsal interosseus, FDI) and lower limb (leg anterior compartment, LEG) muscles. We measured oxidative flux and estimated mitochondrial capacity using the changes in creatine phosphate concentration ([PCr]) and pH as detected by ³¹P magnetic resonance (MR) spectroscopy during isometric exercise and recovery. The highest oxidative ATP flux in sustained exercise was about half the estimated mitochondrial capacity in the LEG (0.38 ± 0.06 vs. 0.90 ± 0.14 mm ATP s⁻¹, respectively), but at the estimated capacity in the FDI (0.61 ± 0.05 vs. 0.61 ± 0.09 mm ATP s⁻¹, respectively). During sustained exercise at a higher contraction rate, intracellular acidosis (pH < 6.88) prevented a rise in oxidative flux in the LEG and FDI despite significantly increased [ADP]. We tested whether oxidative flux could increase above that achieved in sustained exercise by raising [ADP] (> 0.24 mm) and avoiding acidosis using burst exercise. This exercise raised oxidative flux (0.69 ± 0.05 mm ATP s⁻¹) to nearly twice that found with sustained exercise in the LEG and matched (0.65 ± 0.11 mm ATP s⁻¹) the near maximal flux seen during sustained exercise in the FDI. Thus both muscles reached their highest oxidative fluxes in the absence of acidosis. These results show that acidosis inhibits oxidative phosphorylation in vivo and can limit ATP supply in exercising muscle to below the mitochondrial capacity.