Improving accuracy of intrinsic markers and optimizing sampling procedures to estimate fecal output and nutrient digestibility is critical. This experiment was designed to determine the accuracy of indigestible neutral detergent fiber (iNDF) or acid-insoluble ash (AIA) as markers to estimate fecal outputs and nutrient digestibility compared with total collection (TC), and to determine the optimal number of spot samples (SP) to account for diurnal variation in marker and nutrient outputs. The experiment used 12 Holstein cows in a randomized complete block design fed a diet with corn silage or alfalfa silage as the sole forage. During the final 3 d of 21 d periods, total fecal output was collected and 12 spot samples were collected to represent every 2 h in a 24-h cycle. Data were analyzed with a model that included the fixed effect of diet, method or time point, and 2-way interaction and the random effect of cow and cow within block. Estimated fecal outputs and digestibility when using iNDF as a marker were not different from TC, whereas AIA overestimated (P < 0.01) fecal output by 44–61% and underestimated (P < 0.01) DM, OM, and N digestibility by 16–32%. Data from individual SP events were aggregated to represent a sampling frequency of 12 (SP12), 6 (SP6), 4 (SP4) or 2 (SP2) evenly spaced events starting at feeding time. Compared with TC, SP12 did not differ in fecal content of iNDF, OM and N, but fecal AIA content was overestimated (P < 0.01). Comparisons among sampling frequencies with iNDF indicated that, for the corn silage diet, SP6 did not differ from SP12 in fecal iNDF, and N content, whereas SP4 and SP2 over or underestimated (P < 0.01) fecal iNDF and N content compared with SP12. However, fecal iNDF, and N contents did not differ among SP12, SP6, SP4, and SP2 for the alfalfa silage diet. This study confirmed that iNDF was a better fecal marker than AIA to estimate fecal outputs and digestibility, and a spot sampling frequency of at least 6 evenly spaced events starting at feeding time was required for accurate estimation of fecal outputs.