We evaluated whether the dietary roughage-to-concentrate ratio affects ruminal pH and volatile fatty acids (VFAs) in response to a one-time morning fast. Four healthy rumen-cannulated Holstein steers 4-5 months old were used. Cattle were subjected to 2 weeks of adaptation (high-roughage or high-concentrate diet), and morning feed restriction was performed on the day after the adaptation period ended (Day 0). Thereafter, each diet was reintroduced on the evening of Day 0. Our results showed that the 1-hr mean ruminal pH from 0800 to 1900 on Day 0 was higher, and that from 1700 to 1900 on Day 1 was lower (P<0.05) than pH on 1 day before fasting (Day -1) in cattle fed both diets. On Day 0, total VFA levels decreased after morning fasting and were lower (P<0.05) than those on Day -1 irrespective of evening refeeding. Furthermore, blood non-esterified fatty acid and beta-hydroxybutyric acid levels on Day 0 increased and decreased, respectively, compared to Day -1 in cattle fed both diets. These results indicate that even a one-time feed restriction can disrupt ruminal fermentation, and the changes can persist to the next day after fasting.