Three rumen-cannulated Holstein cows in mid to late lactation were randomly assigned to a 3 x 3 Latin square with the following concentrate feeding frequencies: one, two, and four times daily. Cows were fed ad libitum a diet containing 55% corn silage and 45% concentrate (DM basis). The forage portion of the diet was fed twice daily to all cows separately from the concentrate. Each experimental period was 20 d in duration. After a 12-d adaptation, daily individual feed consumption and milk production were measured. Rumen digesta were collected prior to (0 h) and at 2, 4, and 8 h after the morning feeding to measure postprandial changes in protozoa numbers, pH, VFA, and NH3 N. Chromium-EDTA and ytterbium chloride were used as liquid and particulate markers to estimate liquid and solid digesta turnover rate in the rumen. Concentrate and forage DM intake, ruminal protozoa numbers, pH, VFA, and NH3 N concentrations, and digesta turnover rate were not affected by concentrate feeding frequency. There was a significant concentrate feeding frequency with sampling time interaction influence on NH3 N in which feeding concentrate four times daily minimized postprandial fluctuation of rumen NH3 N concentration. Feeding concentrate four times daily increased milk fat and protein production.