The aim of this study was to evaluate the effects on dairy performance and milk fatty acid (FA) composition of (i) supplementation with extruded linseed (EL), (ii) supplementation with synthetic or natural antioxidants, namely vitamin E and plant extracts rich in polyphenols (PERP), (iii) cow breed (Holstein v. Montbéliarde) and (iv) time of milking (morning v. evening). After a 3-week pre-experimental period 24 lactating cows (12 Holstein and 12 Montbéliarde) were divided up into four groups of six cows: the first group received a daily control diet (diet C) based on maize silage. The second group received the same diet supplemented with EL (diet EL, fat level approximately 5% of dietary dry matter (DM)). The third group received the EL diet plus 375 IU/kg diet DM of vitamin E (diet ELE). The fourth group received the ELE diet plus 10 g/kg diet DM of a PERP mixture (diet ELEP). Compared with the diet C, feeding EL-rich diets led to lower concentrations of total saturated FA (SFA) and higher concentrations of stearic and oleic acids, each trans and cis isomer of 18:1 (except c12-18:1), non-conjugated isomers of 18:2, some isomers (c9t11-, c9c11- and t11t13-) of conjugated linoleic acid (CLA), and 18:3n-3. The vitamin E supplementation had no effect on milk yield, milk fat or protein percentage and only moderate effects on milk concentrations of FA (increase in 16:0, decreases in 18:0 and t6/7/8-18:1). The addition of PERP to vitamin E did not modify milk yield or composition and slightly altered milk FA composition (decrease in total saturated FA (SFA) and increase in monounsaturated FA (MUFA)). The minor effects of vitamin E may be partly linked to the fact that no milk fat depression occurred with the EL diet. During both periods the Holstein cows had higher milk production, milk fat and protein yields, and milk percentages of 4:0 and 18:3n-3, and lower percentages of odd-branched chain FA (OBCFA) than the Montbéliarde cows. During the experimental period the Holstein cows had lower percentages of total cis 18:1, and c9,c11-CLA, and higher percentages of 6:0, 8:0, t12-, t16/c14- and t13/14-18:1, and 18:2n-6 than Montbéliarde cows because of several significant interactions between breed and diet. Also, the total SFA percentage was higher for morning than for evening milkings, whereas those of MUFA, total cis 18:1, OBCFA and 18:2n-6 were lower. Extruded linseed supplementation had higher effect on milk FA composition than antioxidants, breed or time of milking.