Aims: To test the hypothesis that the ovarian cycle influences the pressure-pain threshold of the masticatory muscles. Methods: Eighteen healthy women with a regular menstrual cycle (28 ± 2 days), ranging in age from 18 to 35 years, participated in the study. For each subject, pressure-pain thresholds (PPTs) of the masseter and temporalis muscles were assessed at 4 muscular sites by means of an electronic algometer. Measurements were taken at 4 separate sessions across the menstrual cycle corresponding to the following phases: menstrual, follicular, periovulatory, and luteal. Menstrual cycle phases were determined by a pelvic ultrasonographic screening. The study was carried out in a single-blind design, and the initial session was randomly determined for each individual. Data collected were analyzed by repeated-measures analysis of variance. Results: The findings suggest that the PPTs of several masticatory muscles (2 of 4) are influenced by the ovarian cycle, but to a minor extent (P 0.05), and the influence is of limited clinical relevance. Conclusion: In healthy subjects, there is a link between mechanical sensitivity of the masticatory muscles and fluctuation of the ovarian hormones. The relationship between PPTs of the masticatory muscles and the ovarian cycle should be also investigated in patients with temporomandibular disorders and/or orofacial pain conditions.