Muscle fatigue is a reduction in the capacity to exert force and may involve a 'central' component originating in the brain and/or spinal cord. Here we examined whether supraspinal factors contribute to impaired central drive after locomotor endurance exercise. On two separate days, ten moderately active individuals completed a locomotor cycling exercise session or a control session. Brief (2 s) and sustained (30 s) isometric knee extension contractions were completed before and after locomotor exercise consisting of eight, 5 min bouts of cycling at 80% of maximum workload. In the control session, subjects completed the isometric contractions in a rested state. Twitch responses to supramaximal motor nerve stimulation and transcranial magnetic stimulation were obtained to assess peripheral force generating capacity and voluntary activation. Maximum voluntary contraction (MVC) force during brief contractions decreased by 23 ± 6.3 % after cycling exercise, and remained 12 ± 2.8 % below baseline 45 min later (F_1,9 > 15.5, P < 0.01). Resting twitch amplitudes declined by ~45% (F_1,9 = 28.3, P < 0.001). Cortical voluntary activation declined from 90.6 ± 1.6 % at baseline to 80.6 ± 2.1 % after exercise (F_1,9 = 28.0, P< 0.001), and remained significantly reduced relative to control 30-45 min later (80.6 ± 3.4 %, F_1,9 = 10.7, P < 0.01). Thus, locomotor exercise caused a long-lasting impairment in the capacity of the motor cortex to drive the knee extensors. Force was reduced more during sustained MVC after locomotor exercise than in the control session. Peripheral mechanisms contributed relatively more to this force reduction in the control session, whereas supraspinal fatigue played a greater role in sustained MVC reduction after locomotor exercise.