Objective To evaluate the variability of three-dimensional automatic counts of ovarian follicles measuring 2–6 to 2–10 mm during the menstrual cycle and to determine if this test can be applied outside the early follicular phase of the menstrual cycle. Methods: in a prospective observational study, serial transvaginal ultrasound scans were performed from April 20, 2013, to October 30, 2014, on infertile patients. Inclusion criteria: age between 18 and 35 years, BMI 18–25 kg/m (2) , regular menstrual cycles, no history of ovarian surgery and no hormonal changes in TSH, prolactin, fasting insulin or glucose. We excluded patients with ovarian cysts or who did not complete one or more days of the serial transvaginal ultrasound scans. The follicle count was performed in 3D mode ultrasound with a SonoAVC system. Visits were scheduled for the early follicular, mid-follicular, periovulatory and luteal phases of the menstrual cycle. Results Forty-five women were included. The Friedman test showed that the total number of follicles measuring 2–6 mm varied significantly ( p = 0.001) across the four periods of the menstrual cycle. The Paired Student t -test showed a significant increase in 2–6 mm follicle count from the mid-follicular and periovulatory phase to the luteal phase. We found no significant intra-cycle variation between the small follicles (2–6 mm) in the early follicular, mid-follicular and periovulatory phases. The Friedman test showed that the total number of follicles measuring 2–10 mm varied significantly ( p = 0.003) across the menstrual cycle. Conclusions The variation of three-dimensional automatic counts of 2–6 mm follicles in the early follicular, mid-follicular and periovulatory phases was not statistically significant. The significant variability in the counts of follicles measuring 2–10 mm across the menstrual cycle does not permit this examination to be performed side the early follicular phase.