Fibromyalgia (FM) is a musculoskeletal chronic pain syndrome. Its prevalence in women is higher than in men possibly by hormonal factors given that symptoms are aggravated during sex hormone-related events, such as the premenstrual period, pregnancy, postpartum or menopause. The aim of the present study was to investigate whether hyperalgesia and allodynia, in reserpine-induced experimental FM, depend on sex, estrous cycle, ovariectomy and replacement with 17β-estradiol. To fulfill this objective, we compared males, intact females with known estrous cycle phases and ovariectomized (OVX) rats treated with 17β-estradiol. Data demonstrated that reserpine administration disrupted the normal estrous cycle and produced that all females entered metestrus/diestrus. In addition, this treatment leads to muscle hyperalgesia and tactile allodynia in a similar manner in male and intact female rats. However, the absence of ovarian hormones (in OVX rats) increased muscle nociception. 17β-estradiol (2.5–10 μg/rat) produced antihyperalgesic and antiallodynic effects 24 h, but not 8 h, after its administration, suggesting a genomic mechanism. The present results support the validity of the reserpine-induced FM model for searching alternatives of treatment, particularly during endocrine phases when pain is exacerbated such as menopause, and that 17β-estradiol replacement might be useful.