This study examined gender differences in the effect of experimental muscle pain on changes in the relative activation of regions of the upper trapezius muscle during a sustained contraction. Surface electromyographic (EMG) signals were recorded from multiple locations over the upper trapezius muscle with a 10 x 5 grid of electrodes from nine women and nine men during 90 degrees shoulder abduction sustained for 60s. Measurements were performed before and after the injection of 0.4 ml hypertonic (painful) and isotonic (control) saline into the cranial region of the upper trapezius muscle. The EMG root mean square (RMS) was computed for each location of the grid to form a map of the EMG amplitude distribution. The peak pain intensity following the injection of hypertonic saline was greater for women (numerical rating scale 0-10: women 6.0+/-2.1, men 4.2+/-0.9; P<0.01). For both genders, upper trapezius RMS averaged across the grid decreased following the injection of hypertonic saline (P<0.0001). Moreover, there was a relatively larger pain-induced decrease in RMS in the cranial region compared to the caudal region of the muscle for both genders. During the non-painful sustained contractions, the EMG RMS progressively increased more in the cranial than the caudal region, for both men and women, due to fatigue. This mechanism was maintained in men but not in women during the painful condition. The results demonstrate that muscle pain alters the normal adaptation of upper trapezius muscle activity to fatigue in women but not in men.