It is well established that plant oils reduce milk saturated fatty acid content and enhance concentrations of conjugated linoleic acid (CLA) and trans C18:1 in milk fat, but there is increasing evidence to suggest that milk fat CLA responses are often transient and decline over time. It is probable that time dependent adaptations in ruminal biohydrogenation and changes in milk fatty acid composition to lipid supplements are, at least in part, related to the composition of the basal diet. To test this hypothesis, 18 Holstein cows were used in a continuous randomized block design to examine changes in milk fatty acid composition over time in response to plant oils included in diets of variable composition. Cows were randomly allocated to one of three basal diets containing (g/kg dry matter (DM)) maize silage (267) and concentrates (733) (diet C); maize silage (332), grass hay (148) and concentrates (520) (diet M), or grass hay (642) and concentrates (358) (diet H). Basal rations were offered for 21 days, after which diets were supplemented with 50 g sunflower per kg DM (diets C-S and M-S) or 50 g linseed oil per kg DM (diet H-L). Oils were included in all rations incrementally over a five day period (days 0–4), and responses to 50 g/kg DM of the respective oils were evaluated for 17 days (days 4 to 20). Milk fatty acid composition was intensively monitored from days −2 to 20. In contrast to the H-L diet, both C-S and M-S treatments decreased (P<0·05) DM intake, milk fat content and yield, while the C-S diet also reduced (P<0·05) milk yield. Milk fat cis-9, trans-11 CLA and trans-11 C18:1 contents were enhanced on the C-S and M-S treatments but the increases were transient reaching the highest concentrations between days 4 and 6 (cis-9, trans-11 CLA: 1·94 and 2·18 g per 100 g total fatty acids; trans-11 C18:1: 4·88 and 6·23 g per 100 g total fatty acids, respectively) but declined thereafter. In marked contrast, concentrations of cis-9, trans-11 CLA and trans-11 C18:1 in milk from the H-L diet increased gradually over time, responses that were maintained until the end of the experiment (2·89 and 7·49 g per 100 g total fatty acids, respectively).Decreases in milk fat cis-9, trans-11 CLA and trans-11 C18:1 after day 6 on the M-S and C-S diets were associated with concomitant increases in milk fat trans-10 C18:1 content reaching 7·22 and 18·62 g per 100 g total fatty acids on day 18, respectively, whereas concentrations of trans-10 C18:1 in milk on the H-L diet remained low throughout the experiment (0·70 g per 100 g total fatty acids on day 18). Furthermore, milk fat trans-11, cis-13 CLA, trans-11, trans-13 CLA and trans-12, trans-14 CLA contents were all enhanced on the H-L diet, while the M-S and C-S diets increased trans-8, cis-10 CLA, trans-10, cis-12 CLA and trans-9, cis-11 CLA concentrations. Across all diets, decreases in milk fat content were associated with increases in milk trans-10 C18:1, trans-10, cis-12 and trans-9, cis-11 CLA concentrations (r2=0·93, 0·88 and 0·89, respectively). In conclusion, the relative abundance of trans C18:1 and CLA isomers in milk fat were dependent on the composition of the basal diet, type of plant oil and duration of lipid supplementation, highlighting the challenges in developing nutritional strategies for the production of milk highly enriched with CLA over an extended period of time.