There is a significant influence of muscle fatigue on the coupling of antagonistic muscles while patients with post-stroke spasticity are characterized by abnormal antagonistic muscle coactivation activities. This study was designed to verify whether the coupling of antagonistic muscles in patients with post-stroke spasticity is influenced by muscle fatigue. Ten patients with chronic hemipare and spasticity and 12 healthy adults were recruited to participate in this study. Each participant performed a fatiguing isometric elbow flexion of the paretic side or right limb at 30% maximal voluntary contraction (MVC) level until exhaustion while surface electromyographic (sEMG) signals were collected from the biceps brachii (BB) and triceps brachii (TB) muscles during the sustained contraction. sEMG signals were divided into the first (minimal fatigue) and second halves (severe fatigue) of the contraction. The power and coherence between the sEMG signals of the BB and TB in the alpha (8-12 Hz), beta (15-35 Hz), and gamma (35-60 Hz) frequency bands associated with minimal fatigue and severe fatigue were calculated. The coactivation ratio of the antagonistic TB muscle was also determined during the sustained fatiguing contraction. The results demonstrated that there was a significant decrease in maximal torque during the post-fatigue contraction compared to that during the pre-fatigue contraction in both stroke and healthy group. In the stroke group, EMG-EMG coherence between the BB and TB in the alpha and beta frequency bands was significantly increased in severe fatigue compared to minimal fatigue, while coactivation of antagonistic muscle increased progressively during the sustained fatiguing contraction. In the healthy group, coactivation of the antagonistic muscle showed no significant changes during the fatiguing contraction and no significant coherence was found in the alpha, beta and gamma frequency bands between the first and second halves of the contraction. Therefore, the muscle fatigue significantly increases the coupling of antagonistic muscles in patients with post-stroke spasticity, which may be related to the increased common corticospinal drive from motor cortex to the antagonistic muscles. The increase in antagonistic muscle coupling induced by muscle fatigue may provide suggestions for the design of training program for patients with post-stroke spasticity.