BackgroundAcupuncture as a traditional Chinese medicine therapy relies on unique theories to alleviate fatigue. The aim of this study is to evaluate the effect of acupuncture on exercise‐induced fatigue utilizing transcranial magnetic stimulation (TMS).MethodsA total of 20 participants with regular exercise habits were recruited for this study. All participants were randomly assigned to receive either acupuncture or sham acupuncture intervention for exercise‐induced fatigue. TMS and a heart rate monitor were used to measure the amplitude and latency of motor evoked potential (MEP) as well as heart rate every 5 min over a 30‐min period. The blood lactic acid (BLA) levels were measured using Lactate Scout+ at baseline, 0 min, and 30 min after fatigue. Two‐way repeated measures analysis of variance was utilized to compare the differences between the effects of acupuncture method and time. Bonferroni post hoc tests were conducted to compare specific differences. Statistical significance was set at p < .05.ResultsInteraction effect was observed between acupuncture method and time effect in terms of amplitude (F(1, 38) = 5.40, p < .001, η2 = 0.12) and latency (F(1, 38) = 3.78, p = .008, η2 = .09) of MEP. The application of acupuncture can promote the recovery of heart rate especially at 30 min (p < .05), but which seem insufficient to generate significant difference in BLA (F(1, 38) = 0.067, p = .797, η2 = 0.002).ConclusionsAcupuncture can promote the increase of MEP amplitude, shorten MEP latency, and restore heart rate. Preliminary findings provide novel insights for individuals with exercise habits to alleviate fatigue and enhance sports performance.