Follicular fluid (FF) acts as a vehicle for paracrine signalling between somatic cells of the follicle and the oocyte. To investigate changes in the protein composition of FF during ovulation, we conducted a prospective cohort study including 25 women undergoing fertility treatment. Follicular fluid was aspirated either before or 12, 17, 32 or 36?h after induction of ovulation (five patients per time point). Liquid chromatography-mass spectrometry was used to identify and quantify FF proteins. In total, 400 proteins were identified and the levels of 40 proteins changed significantly across ovulation, evaluated by analysis of covariance (adjusted p?<?0.05) and on-off expression patterns. The majority peaked after 12-17?h, e.g., AREG (p?<?0.0001), TNFAIP6 (p?<?0.0001), and LDHB (p?=?0.0316), while some increased to peak after 36?h e.g., ACPP (p?<?0.0001), TIMP1 (p?<?0.0001) and SERPINE1 (p?=?0.0002). Collectively, this study highlights proteins and pathways of importance for ovulation and oocyte competence in humans.