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Background: The effect of time of fecal sampling on the accuracy of acid-detergent insoluble ash (ADIA) and alkaline-peroxide lignin (APL) for the prediction of fecal output (FO) in cattle was evaluated. Eight ruminally cannulated cows (594 ± 35.5 kg) were allocated randomly to 4 bermudagrass [Cynodon dactylon (L.) Pers.] hay diets markedly different in crude protein concentration (79-164 g/kg) with 2 replicates per diet for 3 periods. Cows were offered hay individually at 20 g DM/kg of body weight daily in equal feedings at 08:00 and 16:00 h for a 10-d adaptation period followed by 5-d of total fecal collection. Fecal grab samples also were taken each day during the fecal collection period at 06:00, 12:00, 18:00, and 24:00 h either directly from the rectum or from freshly voided feces. Samples were composited within cow and time across the 5 d total fecal collection period. Additionally, forage, ort, and fecal samples were analyzed for concentrations of APL and ADIA. Results: Fecal concentrations of ADIA and APL were not affected by sampling time (P = 0.22), even though diet affected (P < 0.01) fecal ADIA and APL concentrations. There were no diet × sampling time interactions (P = 0.60). Estimates of FO and dry matter digestibility (DMD) from ADIA and APL were not affected (P = 0.16) by sampling time or the diet × sampling time interaction (P = 0.74). Estimates of FO and DMD from markers from different sampling times or all different combinations of sampling time were not different (P = 0.72) from those of total collection among internal markers. Conclusion: Little variation in concentrations of ADIA and APL in daily fecal excretion across time increases flexibility in fecal grab sampling schedules for predicting FO and DMD. [ABSTRACT FROM AUTHOR]