UNLABELLED: Fibromyalgia syndrome (FMS) is more prevalent in women than in men. The skewed sex distribution in the prevalence has prompted questions of if and how sex hormones may be involved in the pathophysiology of FMS. In this study, we evaluated the levels of sex hormones and pain sensitivity at different phases of a menstrual cycle in regularly menstruating women with FMS relative to age-matched healthy women. Participants (n = 74 in each group) underwent a 9-day urine test to identify the date of ovulation. Three laboratory visits were scheduled to ascertain the varying levels of estrogen (E) and progesterone (P): Late-follicular phase (high E, low P); mid-luteal phase (high E, high P); and perimenstrual phase (low E, low P). At each visit, blood was drawn and ischemic pain testing was performed. The groups did not differ in the fluctuation of luteal hormone, follicular-stimulating hormone, E, and testosterone across a menstrual cycle. FMS patients showed slightly elevated P levels during the mid-luteal phase relative to healthy women but levels were within the normal range. Women with FMS showed consistently lower pain thresholds and tolerance relative to healthy women throughout the menstrual cycle. Pain threshold at the late follicular phase was modestly related to the P level. The results suggest that the disproportionate prevalence of females with FMS is not likely to be attributable to hormonal factors. Furthermore, the role of sex hormones in pain sensitivity for both FMS and healthy women seems to be limited. PERSPECTIVE: Normally menstruating women with FMS and healthy women do not seem to show fluctuating threshold and tolerance to the ischemic pain test. The role of sex hormones in the hyperalgesia of FMS appears limited.