Fatigue post-stroke is a disabling and persistent symptom affecting many stroke survivors. Despite its high prevalence, the pathophysiology underlying this phenomenon remains obscure. The aim of the present study was to investigate the origins of neuromuscular fatigue post-stroke. Ten chronic stroke survivors and 10 controls sustained an isometric contraction at 30% of maximal voluntary contraction (MVC) with the ankle dorsiflexors. Motor evoked potential (MEP), cortical silent period (SP), voluntary activation, M wave and contractile properties were evaluated before, during and after fatigue among the paretic, non-paretic and control limbs. The pattern of response to fatigue in the non-paretic and control limbs was comparable; therefore, results are presented between the paretic and non-paretic limbs. Before fatigue, reduced MVC peak torque and MEP amplitude were observed on the paretic side in comparison with the non-paretic side. During fatigue, the cortical SP duration increased significantly in both limbs, whereas the MEP amplitude significantly increased only in the non-paretic limb. After fatigue, MVC peak torque decreased significantly in both limbs. Significant reductions in M wave and twitch peak torque were observed in both limbs, pointing to the development of peripheral fatigue. However, central fatigue, evident by a significant reduction in voluntary activation, was greater in the paretic than in the non-paretic limb. After stroke, an inability to increase central excitability in response to an increased cortical inhibition associated with the fatiguing contraction may contribute to central fatigue observed in the paretic limb, which may also be linked to increased self-reported fatigue during activities of daily living. These findings advance our understanding of the neuromuscular basis of fatigue post-stroke.