Muscle synergy is the relationship between different muscles in time and space during limb movement, which can make limb motion more stable. It can be applied to the generation of activation patterns in functional electrical stimulation based system and is also important for the assessment of rehabilitation training. Muscle fatigue may affect the muscle synergy, so it is a concern in the rehabilitation applications based on functional electrical stimulation. In order to provide a basis for the subsequent design of muscle synergy-based rehabilitation system, fatigue analysis is first performed on the muscle synergy of six healthy subjects by surface electromyography signal. Considering the complexity of upper limb movements, two forelimb movements, pronation and supination are selected. Then the non-negative matrix factorization algorithm is used to extract the muscle synergy so as to perform the synergistic fatigue analysis among multiple subjects. The experimental results show that when the reconstruction rate reaches the threshold, the complexity of the synergy after fatigue hardly changes, and the repeatability improves to a certain extent, indicating that the fatigue of limb muscles has no negative impact on the synergistic repeatability and complexity. Furthermore, the correlation degree of individual synergy between subjects before and after fatigue is low, which means that the application of muscle synergy in the rehabilitation process of different subjects may need to be personalized adjustment.