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 and arterial oximetry, phosphocreatine (PCr) recovery kinetics and arterial oxygenation were assessed in six 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 ᠰ.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 ᠰ.2 and 1.0 ᠰ.2 min-1 (P < 0.05) as SaO2 was reduced to 82 ᠳ and 77 ᠲ%, 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.