The aim of this study was to investigate whether a significant relation exists between the presence of a negative energy balance (NEB) in cows early in lactation and the reproductive performance after treatment with gonadotrophin-releasing hormone of cows with clinical cystic ovarian disease. Reproductive performance after treatment was assessed from the interval between treatment and first insemination (ITFI) and the interval between treatment and conception (ITC). Based on the outcome of the daily change of milk fat yield (Deltafatg) between the first and second milk recordings post-partum, cows were considered to have passed the NEB nadir (positive Deltafatg) or not (negative Deltafatg). Lactations (n = 430) were divided into four groups according to the interval between calving and first milk recording (ICMR): (i) 0-9 days; (ii) 10-19 days; (iii) 20-29 days; (iv) 30-49 days. The relation between a NEB-proxy parameter (Deltafatg) and reproductive performance was determined. A significant interaction existed for groups 1 and 3 (ICMR on 0-9 and 20-29 days post-partum respectively) with an increased and a decreased probability of being inseminated the first day after treatment (ITFI) respectively. However, no significant interaction was found between Deltafatg and the groups with regard to ITC.