Dairy cows display daily rhythms of milk synthesis that are altered by the time of feed availability and appear to be driven by the circadian clock of the mammary gland. Fatty acids have been shown to entrain circadian rhythms in tissues such as the liver and adipose tissue in experimental models, but their role in the mammary gland is unknown. The objective was to determine the effects of the timing of fatty acid absorption on the daily rhythms of milk synthesis. Nine cannulated lactating Holstein cows (132 ± 90 d postpartum; mean ± SD) were randomly assigned to 1 of 3 treatment sequences in a 3 × 3 Latin square. Treatments were absomasal infusions (132 ± 90 d postpartum; mean ± SD) of 350 g/d of a high C18:1 oil either 24 h/d (CON) or for 8 h/d from 0900 to 1700 h (DAY) or from 2100 to 0500 (NGT). Treatment periods were 12 d with a 5 d washout. During the final 7 d of each period, cows were milked every 6 h to determine the daily patterns milk synthesis across the day. A 24-h rhythm was fit to time-course data using cosine analysis and the amplitude and acrophase (time at peak) were determined. Daily milk, fat, and protein yield and fat and protein concentration were not affected by treatment. Milk yield fit a 24 h rhythm in CON and DAY, and tended to fit a rhythm in NGT. The amplitude of the rhythm of milk yield was increased 29% in DAY compared with CON (P < 0.05), but NGT was not affected. Furthermore, DAY phase delayed daily rhythm of milk yield by 2 h compared with CON and NGT (P < 0.05). Fat and protein concentration exhibited a daily rhythms in CON and NGT (P < 0.05), but not DAY. The amplitude of the rhythm of fat concentration was increased 29% by NGT compared with CON. Fat yield tended to fit a 24 h rhythm in DAY (P = 0.07), but not CON or NGT. Moreover, protein yield fit a 24 h rhythm in all treatments and the amplitude of the rhythm was increased 30% by DAY and decreased 47% by NGT. Fatty acid infusion during the daytime modified the daily rhythms of milk synthesis by increasing the amplitude of milk yield and decreasing the amplitude of fat and protein concentration, whereas infusion at night had little effect.