Abstract Study question Is there a difference in clinical pregnancy rate after single blastocyst transfer (SET) on day 5, in case of stable culture at 36.6 °C or 37.1 °C? Summary answer Clinical pregnancy rates (CPR, heartbeat at 7 weeks) do not differ between blastocyst culture at 36.6 °C and 37.1 °C for five/six consecutive days. What is known already Since the beginning of IVF, embryo culture has been performed at 37.0 °C; however, the optimal temperature remains unknown. Changes in incubator types have led to significant improvements in temperature control. Stable temperature control, with temperature gradients of 0.1 °C among chambers, is possible in G210 incubators (K-Systems, Coopersurgical). A previous prospective trial at our centre showed that embryo development on day 5/6 was not affected when embryos were cultured at a stable temperature of 36.6 °C or 37.1 °C. Though not powered for clinical pregnancy, culture at 37.1 °C resulted in an increased CPR when compared to culture at 36.6 °C (46.4% vs. 74.2%). Study design, size, duration A prospective randomized controlled trial was performed in a tertiary fertility centre between February 2017 and December 2022. Sample size of 89/89 patients with fresh SET was required achieving 80% power to detect a difference of 0.22 between group proportions (0.43-0.65) at a significance level of 0.05 using a two-sided z-test with continuity correction. Participants/materials, setting, methods Patients were recruited the day before oocyte retrieval based on inclusion criteria (SET on day 5, fresh or frozen ejaculated sperm, female age<40 years, BMI<35m2/kg, <3 cycles for the current child) with final randomization after denudation once six mature oocytes were present. Primary endpoint was clinical pregnancy rate (CPR defined as heartbeat at 7 weeks); secondary endpoints were fertilization, blastocyst development, pregnancy (positive hCG), live birth rate (LBR) and cumulative live birth rate (CLBR). Main results and the role of chance A total of 304 patients were eligible for the study of whom 268 signed the consent, 234 were randomized and 181 received SET on day 5: 90 cultured at 36.6 °C and 91 at 37.1 °C. Patients were on average 32.4 ± 3.5 vs. 32.5 ± 4.2 years old, respectively. No differences were observed in embryological outcomes per cycle between both temperatures: 12.0 ± 3.8 vs. 12.1 ± 3.8 COCs retrieved (p = 0.88), 10.0 ± 3.1 vs. 9.7 ± 2.9 mature oocytes inseminated (p = 0.68) with a maturation rate of 83.2% (901/1083) vs. 81.3% (898/1104) (p = 0.87); 8.0 ± 3.1 vs. 7.9 ± 2.7 normally fertilized oocytes with a fertilization rate of 79.9% (720/901) vs. 80.0% (718/898) (p = 0.96), respectively. On average 1.5 ± 1.7 vs. 1.4 ± 1.9 (p = 0.25) and 1.1 ± 1.1 vs. 0.9 ± 1.0 (p = 0.45) blastocysts were vitrified on day 5 and day 6, respectively. Utilization rate per fertilized oocyte was 44.3% (319/720) vs. 42.1% (302/718) (p = 0.14). A single blastocyst transfer was performed for 181 patients, leading to a pregnancy rate of 72.2% (65/90) vs. 62.7% (57/91) (p = 0.17), respectively. CPR of the fresh cycle was 51.1% (46/90) vs. 48.4% (44/91) [OR (95%CI) 1.05 (0.55-1.96), p = 0.71]. To date, a CLBR of 71.1% (64/90) vs. 65.9% (60/91) (p = 0.64), was achieved respectively; with seven patients in each group with remaining blastocysts and no live birth yet. Limitations, reasons for caution Only a selected patient population with expected good prognosis was eligible for the study. Wider implications of the findings Embryos are capable to tolerate small changes in temperature deviations, demonstrated by their similar implantation potential. Trial registration number NCT03548532