Dairy cows have a daily pattern of feed intake which influences ruminal fermentation and nutrient absorption. Milk synthesis also exhibits a daily rhythm and is altered by the timing of feed availability. Nutrients can regulate physiological rhythms, but it is unclear which specific nutrients affect the rhythms of milk synthesis in the cow. The objective of this study was to determine the effect of the timing of acetate infusion on the daily rhythms of feed intake, milk synthesis, milk fatty acids, plasma insulin and metabolites, and core body temperature. Ten lactating ruminally cannulated Holstein cows (127 ± 24.6 d in milk; mean ± standard deviation) were arranged in a 3 × 3 Latin square design. Treatments were ruminal infusions of 600 g/d of acetate either continuously throughout the day (CON) or over 8 h/d during the day (day treatment, DT; 0900 to 1700 h) or the night (night treatment, NT; 2100 to 0500 h). Experimental periods were 14 d with a 7-d washout between periods. Cows were milked every 6 h during the final 7 d of each experimental period to determine the daily pattern of milk synthesis. Blood samples were taken to represent every 4 h across the day and plasma glucose, insulin, β-hydroxybutyrate, urea nitrogen, and acetate concentration were measured. An intravaginal temperature logger was used to measure core body temperature. Data were analyzed with cosinor-based rhythmometry to test the fit of a cosine function with a period of 24 h and to determine the acrophase (time at peak) and amplitude (peak to mean) of each rhythm. Milk yield fit a daily rhythm for all treatments and DT and NT phase-delayed the rhythm and DT increased the robustness of the rhythm. Milk protein concentration fit a daily rhythm for all treatments and DT increased robustness, whereas NT phase-delayed the rhythm. Plasma acetate concentration also fit a daily rhythm in all treatments. Plasma acetate peaked at ~1600 h in CON and DT and at 0053 h in NT, reflecting the timing of treatment infusions. There was a daily rhythm in plasma β-hydroxybutyrate that reflected the plasma acetate rhythm. Core body temperature fit a rhythm for all treatments, but the amplitude of the rhythm was smaller than previously observed. In conclusion, the timing of acetate infusion influences peripheral rhythms of milk synthesis and plasma metabolites.