Maximal oxygen (O ) uptake ( ) is an important parameter with utility in health and disease. However, the relative importance of O transport and utilization capacities in limiting muscle before and after endurance exercise training is not well understood. Therefore, the present study aimed to identify the mechanisms determining muscle pre- and post-endurance exercise training in initially sedentary participants. In five initially sedentary young males, radial arterial and femoral venous (blood samples), leg blood flow (thermodilution), and myoglobin (Mb) desaturation ( H nuclear magnetic resonance spectroscopy) were measured during maximal single-leg knee-extensor exercise (KE) breathing either 12%, 21% or 100% O both pre and post 8 weeks of KE training (1 h, 3 times per week). Mb desaturation was converted to intracellular using an O  half-saturation pressure of 3.2 mmHg. Pre-training muscle was not significantly different across inspired O conditions (12%: 0.47 ± 0.10; 21%: 0.52 ± 0.13; 100%: 0.54 ± 0.01 L min , all q > 0.174), despite significantly greater muscle mean capillary-intracellular gradients in normoxia (34 ± 3 mmHg) and hyperoxia (40 ± 7 mmHg) than hypoxia (29 ± 5 mmHg, both q < 0.024). Post-training muscle was significantly different across all inspired O conditions (12%: 0.59 ± 0.11; 21%: 0.68 ± 0.11; 100%: 0.76 ± 0.09 mmHg, all q < 0.035), as were the muscle mean capillary-intracellular gradients (12%: 32 ± 2; 21%: 37 ± 2; 100%: 45 ± 7 mmHg, all q < 0.029). In these initially sedentary participants, endurance exercise training changed the basis of limitation on muscle in normoxia from the mitochondrial capacity to utilize O to the capacity to transport O to the mitochondria. KEY POINTS: Maximal O uptake is an important parameter with utility in health and disease. The relative importance of O transport and utilization capacities in limiting muscle maximal O uptake before and after endurance exercise training is not well understood. We combined the direct measurement of active muscle maximal O uptake with the measurement of muscle intracellular before and after 8 weeks of endurance exercise training. We show that increasing O availability did not increase muscle maximal O uptake before training, whereas increasing O availability did increase muscle maximal O uptake after training. The results suggest that, in these initially sedentary participants, endurance exercise training changed the basis of limitation on muscle maximal O uptake in normoxia from the mitochondrial capacity to utilize O to the capacity to transport O to the mitochondria.