ObjectiveTo explore the neuromuscular control mechanism of training strategies based on mirror neuron system (MNS): action observation (AO), action execution (AE) and action imitation (AO+AE) using functional Near Infrared Spectroscopy (fNIRS) and surface electromyography (sEMG). MethodsFrom July, 2022 to February, 2023, 64 healthy adults were asked to finish four tasks: watching landscape video (control), watching landscape video and acting right wrist and hand extension (AE), watching right wrist and hand extension video (AO), and watching right wrist and hand extension video and acting right wrist and hand extension (AO+AE). A block design was adopted, five times a task in a block, eight cycles, random orders in videos and tasks. The activation of each channel and regions of interest (ROI, namely BA40, BA44, BA45, BA46, BA6 and BA7) in left MNS regions was detected with fNIRS synchronously, as well as the average electromyography (AEMG) of extensor digitorum and extensor carpi radialis with sEMG. ResultsCompared with the control condition, MNS activated in AO, AE and AO+AE conditions, and the intensities mildly increased in turn. Compared with the control condition, 15 channels activated in AO condition, 15 channels activated in AE condition, and all 20 channels activated in AO+AE condition; and the activation intensities of most channels were AO+AE > AE > AO. Four ROI, BA40, BA46, BA6 and BA7, activated in AO condition, all the six ROI activated in AE and AO+AE conditions, and the activation intensities of most ROI were AO+AE > AE > AO. The standardized AEMG of extensor digitorum and extensor carpi radialis were higher in AO+AE condition than in AE condition (|t| > 4.24, P < 0.001). ConclusionMNS has been activated during action observation, execution and imitation, and the ranges and intensities of activation increase in turn. The target muscles activate more during imitation than during execution. Synchronous application of fNIRS and sEMG is feasible in the study of neural mechanism of rehabilitation strategies based on mirror neuron theory.