Subacute ruminal acidosis (SARA) is one of the most important metabolic disorders, traditionally characterized by low rumen pH, which might be induced by an increase in the dietary proportion of grains as well as by a reduction of structural fiber. Both approaches were used in earlier published experiments in which SARA was induced by replacing part of the ration by a grain mixture or alfalfa hay by alfalfa pellets. The main differences between both experiments were the presence of blood lipopolysaccharide and Escherichia coli and associated effects on the rumen microbial population in the rumen of grain-based induced SARA animals as well as a great amount of quickly fermentable carbohydrates in the grain-based SARA induction experiment. Both induction approaches changed rumen pH although the pH decrease was more substantial in the alfalfa-based SARA induction protocol. The goal of the current analysis was to assess whether both acidosis induction approaches provoked similar shifts in the milk fatty acid (FA) profile. Similar changes of the odd- and branched-chain FA and the C18 biohydrogenation intermediates were observed in the alfalfa-based SARA induction experiment and the grain-based SARA induction experiment, although they were more pronounced in the former. The proportion of trans-10 C18:1 in the last week of the alfalfa-based induction experiment was 6 times higher than the proportion measured during the control week. The main difference between both induction experiments under similar rumen pH changes was the decreasing sum of iso FA during the grain-based SARA induction experiment whereas the sum of iso FA remained stable during the alfalfa-based SARA induction experiment. The cellulolytic bacterial community seemed to be negatively affected by either the presence of E. coli and the associated lipopolysaccharide accumulation in the rumen or by the amount of starch and quickly fermentable carbohydrates in the diet. In general, changes in the milk FA profile were related to changes in rumen pH. Nevertheless, feed characteristics (low in structural fiber vs. high in starch) also affected the milk FA profile and, as such, both effects should be taken into account when subacute acidosis occurs.