Environmental changes and stress may influence passage rate and therefore digestibility in horses participating in a total fecal collection where they are individually housed and fitted with fecal collection harnesses. Existing equine research methods sections frequently do not include descriptions of adaptation periods to allow horses to acclimate to the potential stressors of total fecal collections. Utilization of collection harnesses and confined housing could impact data collection of horse nutrition trials. Therefore, the objective of the current study is to evaluate inter-day variation of dry matter intake (DMI), output (DMO), and resulting calculation of digestibility (DMD) of horses on a 5-d total fecal collection. Twelve mature Quarter horse geldings (13.8 ± 5.6 y; 514.4 ± 38.9 kg) were utilized in a completely randomized design. Horses were fitted with fecal collection harnesses and housed individually in 3.7 m × 3.7 m stalls with ad libitum access to water and mineral block. All horses were fed 1.85% BW coastal bermudagrass hay (AF; 8.92% CP and 33.47% ADF) and 1 kg (AF) of a pelleted concentrate supplement (divided into 2 equal meals) beginning 21 d before the start of total fecal collection and continuing through the 5 d collection. Individual daily intake was recorded, and fecal weights were obtained every 6-h during each day of collection. Subsamples of feed and feces were obtained to determine dry matter using standard AOAC methods. Individual days of collection were separated by all possible pairwise comparisons using PROC MIXED of SAS. Day of collection affected DMO, with increased DMO on d 1 compared with d 3 and 4 (P < 0.05), and a tendency (P ≤ 0.07) for increased DMO on d 1 compared with d 2 and 5. This suggests the start of fecal collection may have altered passage rate to increase DMO. The differences of d 1 DMO affected calculated DMD between days (d 1 and 3, P ≤ 0.04; d 1 and 5, P ≤ 0.09). This highlights the importance of adapting horses to collection techniques before data collection regardless of diet or treatments to be applied, and warrants adequate description within the methods section of research reports.