Isoflavones, phytoestrogens contained in soy foods, may play a role in breast cancer prevention. This randomized double-blinded trial with 34 premenopausal women investigated whether 100 mg of isoflavones per day versus placebo affects the ovulatory cycle during 1 year. Compliance with the study regimen was confirmed by the increase of urinary isoflavone excretion among the intervention group. Blood samples were taken 5 days after ovulation as determined by an ovulation kit, at baseline, and at months 1, 3, 6, and 12. Serum levels of estrone, estradiol, estrone sulfate, progesterone, sex hormone-binding globulin, follicle-stimulating hormone, and luteinizing hormone were quantified by immunoassay; free estradiol was calculated. We applied the method of least squares to fit general linear models to test for an intervention effect while taking into account the repeated measurement design. Except for a small difference in age, the two groups were comparable at baseline. Menstrual cycle length did not change significantly during the intervention [F(1,32) = 0.69; P = 0.44]. During 1 year, we did not observe any significant changes in hormone levels by treatment group. The difference in change between intervention and control group was -13.0 pg/ml (95% confidence interval, -57.5 to 31.5) for estradiol and 6.9 pg/ml (95% confidence interval, -17.8 to 31.5) for estrone. Exclusion of 22 non-ovulatory cycles, noncompliant women, or non-Asian women did not affect the results. These findings do not support the hypothesis that isoflavones affect the ovulatory cycles of premenopausal women over a 1-year period. However, isoflavones alone may have different effects on the reproductive cycle than isoflavones present in soy foods.