Ovarian superstimulation with exogenous follicle-stimulating hormone (FSH) treatment prior to ovum pick-up (OPU) in cattle is common practice to increase in vitro embryo production (IVP) efficiency. The optimal stimulatory period length for OPU-IVP, however, has not been definitively ascertained. The objective of this study, therefore, was to determine the effect of length of the superstimulatory treatment prior to OPU on ovarian response and IVP. Non-pregnant heifers (n=57) 13.8±0.2 months of age and body condition score of 3.0±0.1 (1 to 5 scale) were assigned in a completely randomized design to one of two experimental groups: FSH 2 days 200 mg of FSH (Folltropin-V) distributed in four injections (60, 60, 40, and 40 mg); FSH 3 days 200 mg of FSH distributed in six injections (40, 40, 40, 40, 20 and 20 mg). Injections were performed 12 h apart. Follicular wave emergence was synchronized by dominant follicle removal 36 h prior to the first FSH injection. Intravaginal progesterone (P4) implant (CIDR) was inserted simultaneously to the first FSH injection and removed at OPU, which occurred 44 h after the last FSH injection. Follicle number was determined at OPU and classified as small (<6 mm), medium (6-10 mm) or large (10 mm). Oocytes were classified and subjected to IVP standard procedures. Data were analyzed using generalized linear mixed models (SAS 9.4) and are presented as mean ± SEM. Lengthening the FSH treatment period resulted in greater number of large follicles (FSH2d: 2.5±0.5, FSH3d: 4.5Â±0.6; P=0.01), however, the number of small (5.8±0.5), medium (14.9±1.4) and total follicles (24.2±1.6) was not different between groups. Number of total recovered oocytes (14.9±1.4), viable oocytes (12.8±1.2), cleaved oocytes (7.9±1), as well as recovery rate (59.7%±2.4), percent viable oocytes (86.6%±1.9) and cleavage rate (54.4%±3.9) were not different (P0.2) between groups. Similarly, number of blastocysts produced per heifer (3.2±0.5) and blastocyst rate (21.5±2.7) were not different (P0.9) between groups. In conclusion, lengthening the period of FSH treatment by 1 d increases the number of large follicles at OPU, however, does not improve overall ovarian response, oocyte recovery nor embryo production.