AbstractObjectiveThis study inspects difference of resting state functional connectivity (RSFC) of motor cortex between athletes and ordinary college students and the test‐retest reliability of RSFC.MethodsTwenty high fitness level college students (high fitness group) and 20 ordinary college students (control group) were recruited. The motor cortical blood oxygen signals in resting states were monitored by functional near infrared spectroscopy (fNIRS). RSFCs of brain signals were preprocessed and calculated by FC‐NIRS software. RSFC results of test‐retest reliability were evaluated by intra‐class correlation coefficient (ICC).ResultsTotal RSFC (HbO signal) was significantly different between high fitness group (0.62 ± 0.04) and low fitness group (0.81 ± 0.04) (p< .05). Significant differences were found between the groups (HbO signal) in 50 edges among the 190 edges of motor cortex (14 edges after FDR corrected). At three hemoglobin concentrations, mean of group‐levelICC(C, 1) for total RSFC in two groups was 0.40 ± 0.10, whereas the mean of group‐levelICC(C,k) was 0.57 ± 0.11, depicting “fair” reliability. The mean of group‐levelICC(C, 1) of 190 “edges” was 0.88 ± 0.06, whereas mean ofICC(C,k) was 0.94 ± 0.03, exhibiting “excellent” reliability.ConclusionFitness level is the factor causing specific changes in RSFC strength of motor cortex that can be utilized as biomarker for evaluating the fitness level.