Aims: To investigate the local and segmental effects of therapeutic ultrasound at a dose of 0.4 w/cm2 with 100% duty cycle for 5 minutes compared to the effect of sham ultrasound on painful masticatory muscles. Methods: A total of 20 adult female subjects with bilateral masseter myalgia diagnosed according to the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) were included. Each subject was randomized to either an active ultrasound group or a sham ultrasound group. The intervention was applied to each masseter muscle for 5 minutes. Measures included pre- and post-self-reported pain intensity recorded on a verbal rating scale (VRS), pressure pain thresholds for the masseter (PPT-M) and temporalis (PPT-T) muscles, and intraoral temperature for the masseter muscle. Preintervention score was subtracted from the postintervention score for all measures to calculate mean change in pain, and nonparametric Mann-Whitney test was used to compare the groups. Statistical significance was set at P .05. Results: Changes in VRS did not show a significant difference between groups (P > .05). There were significant increases in PPT-M and intraoral temperature in the ultrasound group compared to the sham group (P .05). There was no significant difference in PPT-T (P > .05), suggesting no segmental effect. Conclusion: Therapeutic ultrasound produced an immediate increase in PPT-M and intraoral temperature compared to sham ultrasound in female subjects with bilateral masseter myalgia. Keywords: myalgia, TMD, ultrasound