An important aspect of central fatigue is supraspinal fatigue, or fatigue originating from an insufficient output from the motor cortex. One possible underlying mechanism is that this reaction is evoked by changes in brain neurotransmitters such as dopamine (DA) and noradrenaline (NA). In the present study, we looked into the relation between supraspinal fatigue and changes in brain neurotransmitter concentrations before and after prolonged exercise. Ten well-trained male cyclists participated in this study. Subjects exercised in 18°C and performed 60 min at 55% Wmax followed by a time trial which required the subject to complete a work equal to 30 min at 75% Wmax as quickly as possible. Pharmacological interventions were placebo, methylphenidate (DA reuptake inhibitor) and reboxetine (NA reuptake inhibitor). Voluntary activation and corticospinal excitability changes were tested in the knee extensors using TMS and motor nerve electrical stimulation before and after the cycling exercise. Reaction time and attention were measured using the psychomotor vigilance test (PVT). NA administration decreased performance significantly (9%). This was accompanied by a significant reduction in voluntary activation. No differences were observed for DA reuptake inhibition. No changes in corticospinal excitability were observed. The PVT revealed that reaction time was negatively influenced by reboxetine. Higher NA concentrations in the brain enhance central fatigue. The reduction in both the voluntary activation and reaction time shows that this decrease in performance was centrally mediated.