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Abstract Background Skeletal mandibular protrusion would influence to the muscle fatigue of the masticatory muscles. Establishing a diagnostic procedures combining physiological and biochemical information is necessary for quantitative evaluation of masticatory muscle fatigue. Objective The transverse relaxation time (T2 time) of muscle functional magnetic resonance imaging (mfMRI), and 31P‐magnetic resonance spectroscopy (MRS) were used to investigate the reliability as parameters for measuring the masseter muscle in patients with skeletal mandibular prognathism. Method The subjects were 19 patients diagnosed as skeletal mandibular protrusions and 19 healthy subjects as a control group. Transverse relaxation time (T2 value) determined by mfMRI along with creatine phosphate (PCr) and inorganic phosphorus (Pi) determined by 31P‐MRS before, during, and after clenching were used for molecular imaging of muscle fatigue. Results The average T2 value of the patient group was significantly higher than that of the healthy control group at rest. Furthermore, the average T2 value transiently increased in both groups during experimental clenching. The PCr and Pi showed a tendency toward a transient decrease and increases, respectively. The pH in the masseter muscle showed a transient decrease in both groups prior to and following experimental clenching. The pH in the masseter muscle of the patient group was significantly lower than that in the healthy control group at rest and recovery. Conclusion We showed mfMRI and 31P‐MRS are useful for evaluating masseter fatigue during clenching, and the masseter muscle in the prognathic patients showed more severe fatigue than the healthy controls.