Background: According to several laboratory protocols and specific conditions, in vitro fertilization (IVF) dishes with culture media can be prepared 24 hr in advance compared to routine protocols. However, it is not clear if this procedure can affect embryological outcomes. Methods: A nested case-control study was done in a cohort of couples undergoing IVF at the Infertility Unit of the ASST Lariana from August 2016 to July 2018. Cases were patients undergoing ovum pick up after a laboratory day off. Controls were patients undergoing ovum pick up after working days from Monday to Thursday. Culture media for oocyte culture and insemination were prepared about 42 and 18 hr before oocyte retrieval for cases and controls, respectively. Cases and controls were matched with a 1:2 ratio (for age, inseminated oocytes, length of stimulation). The "Good-Quality-Index" (GQI) was the main outcome to be compared between the two groups and was defined as good quality transferred or cryopreserved embryos on day 2 or 3+number of good quality blastocysts/inseminated oocytes. Results: A total of 76 cases and 152 matched controls were enrolled. The median GQI was equal to 33.0% (IQR: 20.0-50.0%) and 33.0% (IQR: 25.0-50.0%), in cases and controls, respectively (p=0.40). Study groups and GQI were not significantly correlated (correlation coefficient r=0.047, p=0.48). Main embryological parameters and cumulative pregnancy rates were similar between the two groups. Conclusion: Our data support the vision that culture media can be prepared 24 hr in advance compared to routine protocols without affecting embryological outcomes.