<p>Background: According to several laboratory protocols and specific conditions, <em>in vitro </em>fertilization (IVF) dishes with culture media can be prepared 24 <em>hr</em> in advance compared to routine protocols. However, it is not clear if this procedure can affect embryological outcomes.&nbsp;<br /> 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 <em>hr</em> 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 &quot;Good-Quality-Index&quot; (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.<br /> 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.<br /> Conclusion: Our data support the vision that culture media can be prepared 24 <em>hr</em> in advance compared to routine protocols without affecting embryological outcomes.</p>