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The aim of this study was to determine the individual and combined effects of supplementing fat with whole-cracked rapeseed (FAT), nitrate (NITRATE), and 3-nitrooxypropanol (3-NOP) on compositional and functional properties of milk fat. An 8 × 8 incomplete Latin square design was conducted with 48 lactating Danish Holstein cows over 6 periods of 21 d each. Eight diets were 2 × 2 × 2 factorially arranged: FAT (30 or 63 g crude fat/kg DM), NITRATE (0 or 10 g nitrate/kg DM), and 3-NOP (0 or 80 mg 3-NOP/kg DM), and cows were fed ad libitum. Milk samples were analyzed for general composition, fatty acids (FA) and thermal properties of milk fat. Milk fat content was decreased by supplementing fat but increased by 3-NOP. The changes in FA composition were mainly driven by the FAT × 3-NOP interaction. Fat supplementation shifted milk FA composition toward a lower content of SFA and greater contents of MUFA and PUFA, whereas these effects became smaller in combination with 3-NOP. However, 3-NOP had no effects on SFA, MUFA, or PUFA in low-fat diets. Fat supplementation lowered solid-fat content in milk fat because of the decreased SFA content. The onset crystallization temperature of milk fat was decreased by 3-NOP when supplemented in low-fat diets. According to the FAT × 3-NOP interaction, supplementation of fat without 3-NOP shifted the peak temperature of the low-melting fraction of milk fat toward low temperature as a result of a decreased proportion of C16:0 and increased proportions of C18:1 cis-9, C18:1 trans-11, C18:2 cis-9, and CLA cis-9,trans-11. In conclusion, no additive effects were observed among FAT, NITRATE, and 3-NOP on chemical and thermal properties of milk fat, and fat supplementation largely changed milk FA composition, which in turn affected the thermal properties of milk fat., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)