OBJECTIVE: To investigate the effects of muscle fatigue induced by low-level isometric jaw-clenching and subsequent glutamate-evoked muscle pain on the exteroceptive suppression (ES) response and resting electromyographic (EMG) activities in human jaw muscles. METHODS: The resting EMG activity and the ESs were recorded before (baseline), after low-level jaw-clenching (Post1), after subsequent glutamate or isotonic saline injections into the left masseter (Post2), and 60 min after the clenching (Post3) in 23 healthy volunteers. RESULTS: The late ES (ES2) showed more inhibition at Post1 compared with baseline (P<0.05). It was less inhibited after both types of injections (Post2), and increased at Post3 again (P<0.05) with no significant difference between the glutamate and isotonic saline sessions. The resting EMG activity increased at Post1 and Post2 (P<0.05). The glutamate injection further increased the resting EMG activity in the injected muscle (P<0.01). CONCLUSIONS: Muscle fatigue influences inhibitory reflex pathways in jaw-closing muscles and subsequent acute muscle pain potentiates the local increase in the resting EMG activity of the painful muscle. SIGNIFICANCE: Muscle fatigue which can be observed in patients with oral dysfunctions may interact with nociceptive regulation and influence the clinical presentation of jaw symptoms and function.