The cow’s daily pattern of feed intake creates differences in nutrient consumption across the day. Feeding a high-fiber diet during the high-intake period of the day is a potential strategy to stabilize rumen fermentation and nutrient absorption. Rumen samples from a previously published experiment (Rottman et al., J. Dairy Sci. 98:7), which fed multiple diets differing in neutral detergent fiber and starch across the day, were used to determine the effects on the daily patterns of selected microbial populations. Briefly, diets included a control (33.3% NDF), a low fiber diet (LF; 29.6% NDF), and a high-fiber diet (HF; 34.8% NDF). Nine cannulated Holstein cows were fed 1 of 3 diet combinations in a 3 × 3 Latin square design: (1) 100% daily offering of the control diet at 0900 h (CON); (2) 70% daily offering of HF at 0900 h and 30% daily offering of LF at 2200 h (HL); or (3) 30% daily offering of LF at 0900 h and 70% daily offering of HF at 1300 h (LH). Microbial DNA was extracted from rumen digesta representing every 3 h across the day. The relative abundances of bacteria, fungi, ciliate protozoa, Fibrobacter succinogenes, Ruminococcus albus, Butyrivibrio fibrisolvens, Selenomonas ruminantium, Butyrivibrio hungatei, Prevotella bryantii, Megasphaera elsdenii, and Streptococcus bovis was measured using quantitative PCR. Data were analyzed using the mixed procedure of SAS with a repeated statement and tested the effects of treatment, time, and their interaction. The time of day affected the relative abundance of all microbial groups (P < 0.05) and nearly all were modified by feeding strategy. Notably, HL and LH treatments caused dramatic increases in the relative abundances of S. bovis (4-fold), S. ruminantium (2-fold), and B. hungatii (~3-fold) at 0900 h, that was not observed in the control (P < 0.05). Total bacteria abundance varied by treatment, and the lowest daily variation occurred in HL. Results suggest that the daily pattern of certain microbies is modified by altering the amounts of fiber and starch fed throughout the day, and feeding a high-NDF diet during peak intake may stabilize bacterial abundance.