Supraspinal fatigue, defined as an exercise-induced decline in force caused by suboptimal output from the motor cortex, accounts for over one-quarter of the force loss after fatiguing contractions of the knee extensors in normoxia. We tested the hypothesis that the relative contribution of supraspinal fatigue would be elevated with increasing severities of acute hypoxia. On separate days, 11 healthy men performed sets of intermittent, isometric, quadriceps contractions at 60% maximal voluntary contraction to task failure in normoxia (inspired O2 fraction/arterial O2 saturation = 0.21/98%), mild hypoxia (0.16/93%), moderate hypoxia (0.13/85%), and severe hypoxia (0.10/74%). Electrical stimulation of the femoral nerve was performed to assess neuromuscular transmission and contractile properties of muscle fibers. Transcranial magnetic stimulation was delivered to the motor cortex to quantify corticospinal excitability and voluntary activation. After 10 min of breathing the test gas, neuromuscular function and cortical voluntary activation prefatigue were unaffected in any condition. The fatigue protocol resulted in ∼30% declines in maximal voluntary contraction force in all conditions, despite differences in time-to-task failure (24.7 min in normoxia vs. 15.9 min in severe hypoxia, P <0.05). Potentiated quadriceps twitch force declined in all conditions, but the decline in severe hypoxia was less than that in normoxia (P <0.05). Cortical voluntary activation also declined in all conditions, but the deficit in severe hypoxia exceeded that in normoxia (P <0.05). The additional central fatigue in severe hypoxia was not due to altered corticospinal excitability, as electromyographic responses to transcranial magnetic stimulation were unchanged. Results indicate that peripheral mechanisms of fatigue contribute relatively more to the reduction in force-generating capacity of the knee extensors following submaximal intermittent isometric contractions in normoxia and mild to moderate hypoxia, whereas supraspinal fatigue plays a greater role in severe hypoxia.