Inadequate cerebral O₂ availability has been proposed to be an important contributing factor to the development of central fatigue during strenuous exercise. Here we tested the hypothesis that supraspinal processes of fatigue would be increased after locomotor exercise in acute hypoxia compared to normoxia, and that such change would be related to reductions in cerebral O₂ delivery and tissue oxygenation. Nine endurance-trained cyclists completed three constant-load cycling exercise trials at ∼80% of maximal work rate: (1) to the limit of tolerance in acute hypoxia; (2) for the same duration but in normoxia (control); and (3) to the limit of tolerance in normoxia. Throughout each trial, prefrontal cortex tissue oxygenation and middle cerebral artery blood velocity (MCAV) were assessed using near-infrared spectroscopy and trans-cranial Doppler sonography, respectively. Cerebral O₂ delivery was calculated as the product of arterial O₂ content and MCAV. Before and immediately after each trial, twitch responses to supramaximal femoral nerve stimulation and transcranial magnetic stimulation were obtained to assess neuromuscular and cortical function, respectively. Exercise time was reduced by 54%in hypoxia compared to normoxia (3.6 ± 1.3 vs. 8.1 ± 2.9 min; P<0.001). Cerebral O₂ delivery,cerebral oxygenation and maximum O₂ uptake were reduced whereas muscle electromyographic activity was increased in hypoxia compared to control (P <0.05).Maximum voluntary force and potentiated quadriceps twitch force were decreased below baseline after exercise in each trial;the decreases were greater in hypoxia compared to control (P<0.001), but were not different in the exhaustive trials (P>0.05). Cortical voluntary activation was also decreased after exercise in all trials, but the decline in hypoxia (Δ18%) was greater than in the normoxic trials (Δ5-9%)(P <0.05). The reductions in cortical voluntary activation were paralleled by reductions in cerebral O₂ delivery. The results suggest that curtailment of exercise performance in acute severe hypoxia is due, in part, to failure of drive from the motor cortex, possibly as a consequence of diminished O₂ availability in the brain.