Abstract Study question What is the best follicle size, estradiol (E2) serum concentration and endometrial thickness to trigger ovulation in natural cycles? Summary answer Optimal follicles size is 18–22mm but estrogen concentration also need to be considered to maximize oocyte maturity and to minimize premature LH surge. What is known already Timing of the ovulation triggering is essential in infertility treatments based on natural menstrual cycles such as optimized vaginal intercourse, intrauterine inseminations and thawing cycles without hormone replacement therapy. Common parameters to define the day of ovulation triggering are the follicle size and the estrogen concentration. However, data on follicle size and estrogen concentration are either derived from longitudinal evaluations of few ideal participants, are not very detailed or were studied in stimulated cycles. The model of Natural Cycle IVF (NC-IVF) which provides more detailed information has never been used to study this issue. Study design, size, duration Retrospective cross sectional analysis of monofollicular NC-IVF cycles. Follicle size, E2 and LH serum concentrations and endometrial thickness were evaluated on day –5 to 0 (day 0 = day of aspiration). Ovulation was triggered with 5.000IE HCG 36h before aspiration if follicle size was 14–22mm. Patients with irregular cycles, endometriosis >II°, cycles with azoospermia or cryptozoospermia and with inconsistent data were excluded. 606 cycles from 290 women were analysed from 2016 to 2019. Participants/materials, setting, methods Mean age of women undergoing NC-IVF was 35.8±4.0y, median 36y [IQ-range: 34;39]. Each woman performed mean 2.1±1.4, median: 2 [IQ-range: 1–3] NC-IVF cycles at an university based IVF center. All parameters were analysed inter and intraindividually and associations were adjusted for maturity of oocyte, zygote development rate, embryo score, implantation rate and live birth rate. Associations were adjusted for age, cause of infertility and number of previous transfers. Main results and the role of chance Follicle size, E2 concentration and endometrial thickness increased constantly over time. The increase was computed for each cycle without considering any correlation intra patient, revealing an increase of follicle size by 1.04±0.64mm, an increase of E2 concentration by 167.3±76.8pmol/L and endometrial thickness by 0.69±0.59mm per day. Based on a multivariate adjusted model with follicle size, E2 and their interaction, number of retrieved oocytes was associated with E2 concentration (aOR 1.80, 95% CI 1.05–3.11; p = 0.034). Maturity of oocytes was associated not only with E2 concentration (aOR 1.84, 95% CI 1.15–2.94; p = 0.010) but also with follicle size (aOR 1.24, 95% CI 1.01–1.53; p = 0.037) and so was also the interaction of both parameters (aOR 0.96, 95% CI 0.94–0.99; p = 0.017). LH surge was calculated to start in 25% of cases at an E2 level of 545 pmol/l, in 50% of cases at 907pmol/l and in 75% of cases at an E2 level of 1531pmol/l. Live birth rate in cycles with follicles size 14–17 mm was 2.2–3.5% per initiated cycle and in cycles with follicle size 18–22mm 8.5–12.5%. Limitations, reasons for caution Cross sectional studies provides less precise information than longitudinal studies. Follicle size and endometrial thickness were evaluated by several physicians possibly causing some imprecision. Wider implications of the findings: There is a trend towards natural treatment cycles. The study contribute to an optimisation of infertility treatments involving natural cycles. The study gives guidance about the number of days required after a follicle monitoring to reach the optimal time for triggering ovulation. Trial registration number Not applicable