In contrast to their exercise trained counterparts, the maximal oxidative rate of skeletal muscle in sedentary humans appears not to benefit from supplemental O₂ availability but is impacted by severe hypoxia, suggesting a metabolic limitation either at or below ambient O₂ levels. However, the critical level of O₂ availability at which maximal metabolic rate is reduced in sedentary humans is unknown. Using ³¹P magnetic resonance spectroscopy (MRS) and arterial oximetry, PCr recovery kinetics and arterial oxygenation were assessed in 6 sedentary subjects performing 5 min bouts of plantar flexion exercise followed by 6 min of recovery. Each trial was repeated while breathing one of four different fractions of inspired O₂ (FIO₂) (0.10, 0.12, 0.15, and 0.21). The PCr recovery rate constant (a marker of oxidative capacity) was unaffected by reductions in FIO₂, remaining at a value of 1.5 ± 0.2 min^-1 until arterial O₂ saturation (SaO₂) fell to less than ~ 92%, the average value reached breathing an FIO₂ of 0.15. Below this SaO₂, the PCr rate constant fell significantly by 13 and 31% to 1.3 ± 0.2 and 1.0 ± 0.2 min^-1 (P<0.05) as SaO₂ was reduced to 82 ± 3 and 77 ± 2 %, respectively. In conclusion, this study has revealed that O₂ availability does not impact maximal oxidative rate in sedentary humans until the O₂ level falls well below that of ambient air, indicating a metabolic limitation in normoxia.