The forage:concentrate ratio affected only a few fatty acids at 35 DIM; most notably, cows fed the high-forage diets had higher concentrations of 16:0 and lower concentrations of trans-10 18:1 and 18:2. At 75 DIM, the high-forage diet reduced concentrations of 18:2 and increased concentrations of 4:0. At 125 DIM, feeding the high-forage diet generally increased concentrations of SCFA and 16:0 and reduced concentrations of LCFA.