This research communication hypothesizes that superstimulation with follicle-stimulating hormone (FSH) would not impact behavior and performance of Holstein cows. The objectives were to investigate the effect of FSH superstimulation on follicular dynamics, animal behaviour, body surface temperature and milk yield of Holstein cows. Cows were blocked by parity and body condition score (BCS), and within a block, they were assigned randomly to receive either normal saline (CON = 7) or 500 IU of FSH (n = 8). The estrous cycle of cows was synchronized by using two injections of prostaglandin F2-alpha (PG), 11 days apart. The dominant follicle was ablated at the time of the second PG injection, and an intra-vaginal controlled internal drug release (CIDR) was inserted (day 0). Two days later, FSH treatment was initiated and continued for 3 days in six equal doses of 83.33 IU after 12 h. Follicles were counted and their sizes were measured from day-0 to day-5. Behavior, including activity and feeding time, was recorded using SmartTag Neck from day 0 to 6. The surface temperatures of the eye, shoulder, flank, and vulva were measured by using infrared thermal imaging every 12 h from day-2 to day-5. Milk was recorded from day-0 to day-6. Mixed effects models were used to analyse the data using SAS statistical software. The number of small and medium follicles did not differ between treatments. However, FSH-treated cows had a more (P = 0.01) large and total follicles compared with CON cows. FSH treatment did not affect activity, feeding time, body surface temperature, or milk yield. In conclusion, FSH superstimulation increased the number of large follicles but did not influence behaviour, body surface temperature, or performance in dairy cows.