This study aims to evaluate the acupuncture effect on exercise-induced fatigue utilizing transcranial magnetic stimulation (TMS). A total of 20 sports enthusiasts were recruited in this study, all participants would be assigned to receive acupuncture and sham acupuncture intervention on exercise-induced fatigue randomly. TMS and heart rate monitor would be used to measure the amplitude and latency of motor evoked potential (MEP) and heart rate every 5 minutes over a 30-minute period. Lactate Scout+ was used to measure the blood lactic acid (BLA) in baseline, 0 minutes and 30 minutes after fatigue. Two-way repeated measures ANOVA was used to compare the difference between acupuncture method and time effects. Bonferroni post hoc tests were conducted to compare specific differences. Significance was set at P < 0.05. Interaction effect was found between acupuncture method and time effect in amplitude (F (1, 38) = 5.40, P < 0.001, η2 = 0.12) and latency (F (1, 38) = 3.78, P = 0.008, η2 = 0.09) of MEP. Acupuncture intervention can promote the recovery of heart rate especially at 30 minutes (P < 0.05), but which seem insufficient to generate significant difference in BLA (F (1, 38) = 0.067, P = 0.797, η2 = 0.002). Preliminary findings provide new perspectives for sports enthusiasts to relieve fatigue and reduce the inhibition of motor cortex excitability utilizing acupuncture.