Twelve ruminally cannulated cows (120 ± 52 DIM; 35.5 ± 8.9 kg of milk/d; mean ± SD) were randomly assigned to treatment in a Latin square design with 21-d periods. Treatments were (1) subacute ruminal acidosis (SARA) induction, (2) recovery, and (3) control. SARA was induced by feeding a diet containing 29% starch, 24% NDF, and 2.8% fatty acids (FA), whereas the recovery and control diets contained 20% starch, 31% NDF, and 2.3% FA. Experimental sampling took place on d 0, 3, 7, 10, 14, 17, and 21 of each period. Data were analyzed as repeated measures using the MIXED procedure of SAS. Dry matter intake and milk yield were increased during induction of SARA from d 10 to 21 and from d 14 to 21, respectively (P < 0.05). Milk fat concentration was reduced from d 3 to 14 of SARA induction, whereas lower milk fat yield was observed on d 3 only (P < 0.05). The acetate-to-propionate ratio was lower, whereas the concentrations of total volatile FA and lactate were greater during SARA as compared with the control period (P < 0.05). Plasma insulin and lipopolysacharide binding protein concentrations increased during SARA induction, whereas plasma nonesterifed FA and milk BHBA decreased (P < 0.05). The ratio of milk t10 to t11 18:1 increased during the SARA induction period (P < 0.05), but concentration of t10 18:1 remained below 0.5% of FA, whereas t10c12 18:2 was not detected. The ratio of t10-to-t11 C18:1 was positively correlated with the acidosis index (area under pH <5.8/DMI; r = 0.36; P < 0.05). Negative associations were observed between the acidosis index and the milk fat concentrations of iso 14:0, iso 15:0, iso 16:0, iso 17:0, and anteiso 17:0 (r = −0.23 to −0.30, P < 0.05). Plasma cytokines (IFNα, IFNγ, IL-13, IL-1α, IL-1 F5, IL-21, IP-10, MIG, MIP-1β, and TNFα) were not affected by treatment at any time point. From d3 to 21, most parameters were not different between the recovery and the control periods. Induction of SARA resulted in a rapid and transient reduction in milk fat synthesis, which was not strongly associated with ruminal biohydrogenation.