Although phosphorus magnetic resonance spectroscopy ((31)P-MRS)-based evidence suggests that in vivo peak mitochondrial respiration rate in young untrained adults is limited by the intrinsic mitochondrial capacity of ATP synthesis, it remains unknown whether a large, locally targeted increase in convective O2 delivery would alter this interpretation. Consequently, we examined the effect of superimposing reactive hyperemia (RH), induced by a period of brief ischemia during the last minute of exercise, on oxygen delivery and mitochondrial function in the calf muscle of nine young adults compared with free-flow conditions (FF). To this aim, we used an integrative experimental approach combining (31)P-MRS, Doppler ultrasound imaging, and near-infrared spectroscopy. Limb blood flow [area under the curve (AUC), 1.4 ᠰ.8 liters in FF and 2.5 ᠰ.3 liters in RH, P < 0.01] and convective O2 delivery (AUC, 0.30 ᠰ.16 liters in FF and 0.54 ᠰ.05 liters in RH, P < 0.01), were significantly increased in RH compared with FF. RH was also associated with significantly higher capillary blood flow (P < 0.05) and faster tissue reoxygenation mean response times (70 ᠱ5 s in FF and 24 ᠱ5 s in RH, P < 0.05). This resulted in a 43% increase in estimated peak mitochondrial ATP synthesis rate (29 ᠱ3 mM/min in FF and 41 ᠱ4 mM/min in RH, P < 0.05) whereas the phosphocreatine (PCr) recovery time constant in RH was not significantly different (P = 0.22). This comprehensive assessment of local skeletal muscle O2 availability and utilization in untrained subjects reveals that mitochondrial function, assessed in vivo by (31)P-MRS, is limited by convective O2 delivery rather than an intrinsic mitochondrial limitation.