We evaluated the proportion of fatty acid groups, with an emphasis on hypercholesterolaemic fatty acids, in the milk of 25 Holstein cows during the 1(st) period of lactation in relation to their negative energy balance (NEB). Sampling of each cow's milk started on the 7th day after calving. Milk samples (n = 425) were collected at 7-day periods during the first 17 weeks of lactation. The proportion (%) of saturated (SFA), hypercholesterolaemic (HCFA), volatile (VFA), unsaturated (UFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids in the milk fat was determined. Body condition score and fat to protein ratio in milk were applied for precise determination of the NEB breakpoint during the observed period. The effects of parity, NEB, regression on lactation week and fat to protein ratio were evaluated using SAS 9.3. Milk contained a lower proportion of SPA as well as equally higher UFA (+/- 2.13%; P < 0.01) during the NEB period. The overcoming of NEB caused an increase in SEA, however, and simultaneously a significant decline in total HCFA (-1.86%; P < 0.01) as well as main MUFA (-1.81%, P < 0.05). The results document the necessity of increasing Holstein cow robustness to meet the production conditions in dairy farms in relation to the requirement of higher nutrient quality as well as the potential health benefits of cow's raw milk for consumers.