The purpose of this study was to investigate the effects of jaw-muscle fatigue evoked by low-level tooth-clenching followed by the induction of experimental muscle pain by injection of glutamate on the perception of fatigue and pain and on the resting electromyographic (EMG) activity. In addition, the role of gender on these interactions was studied. The EMG activities of bilateral masseter (MAL, MAR) and temporalis (TAL, TAR) muscles in 11 healthy young women and 12 men were measured before (Baseline) and after tooth-clenching for 30 min at 10% of maximal force (Post1), after subsequent glutamate (Glu) or isotonic saline (Iso) injection into the MAL following the tooth-clenching (Post2) and 60 min after tooth-clenching (Post3). The intensities of fatigue, fatigue-related muscle pain and headache-like symptoms were scored on 0-10 cm visual analog scales (VAS). The glutamate-evoked pain was continuously scored on an electronic VAS. Sustained low-level tooth-clenching consistently produced fatigue sensation, fatigue-related muscle pain and headache-like symptoms in both genders with significantly higher fatigue VAS scores in men than in women, while the accompanying increase in the resting EMG activity appears higher in women than in men in the masseter muscles. In this study no gender differences were found for the perceived amount of experimental pain induced by glutamate injection. Additional increases of the resting EMG activity after injections occurred only in men in the injected masseter muscle and non-injected temporalis muscles. The present findings provide new information on the complex influence of gender on sensory-motor integration in the trigeminal system which may contribute to differences in susceptibility to develop musculoskeletal pain problems.