Objective The aim of this study was to evaluate whether values of FSH, LH, estradiol, anti-Müllerian hormone (AMH), inhibin B, antral follicle count (AFC) and ovarian volume (OV) determined on day 7 of the hormone-free interval are similar to values measured on days 2-5 of two subsequent natural menstrual cycles. In addition, values measured on day 7 of the hormone-free interval were examined for the purpose of predicting values measured on days 2-5 during the second natural cycle. Methods In this study, 25 women using hormonal contraception provided a blood sample and underwent transvaginal ultrasound measurements on day 7 of the hormone-free interval and on cycle days 2-5 of two subsequent natural cycles. Changes were compared by repeated measures ANOVA and multivariate linear regression was used for prediction purposes. Result SMean (SD) age of the participants was 26.3 (6.2) years. Overall significant decreases in FSH and inhibin B and significant increases in AMH, AFC and ovarian volume values were measured after discontinuation of hormonal contraception (P < 0.001, P = 0.04, P = 0.01, P < 0.001 and P = 0.004, respectively). Significant changes occurred both from day 7 of the hormone-free interval to natural cycle 1 as well as from natural cycle 1 to natural cycle 2. FSH, AMH and AFC values measured during days 2-5 of natural cycle 2 could be predicted by the corresponding values measured on day 7 of the hormone-free interval. CONCLUSION Hormonal and ultrasound markers of ovarian function in hormonal contraception users measured at the end of the hormone-free interval do not seem to represent subsequent natural early follicular phase values. However, these values can, in some cases (FSH, AMH and AFC), be used to predict early follicular phase values using calculated prediction equations, which need to be validated in future research. © The Author 2010.