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Non-invasive endocrinology is an important tool for animal conservation, but its success depends on many factors (e.g. adequate hormonal extraction, diet, antibody used in the assay). Dietary fiber is one of the main sources that can lead to erroneous interpretation of the endocrine status provided by EIA analysis. This study aimed to evaluate the dietary fiber effect on the fecal androgen metabolites (FAM) detection, on the daily defecation rate and fecal production, as well as to analyze the gastrointestinal passage and retention time of the experimental diets. Eight brown brocket deer (Mazama gouazoubira) males were randomly assigned to two groups and submitted to both isocaloric and isoproteic experimental diets for 10 days, in a crossover system: low fiber percentage feed (LF, 7% fiber) and high fiber percentage feed (HF, 19% fiber). Such groups were alternated in the middle of this period, with an interval of 10 days between them. In addition, there was a five-day adaptation phase at the beginning of each diet. Fecal collection for FAM measurement was performed during 10 days of treatment, whereas,the defecation rate and fecal production were performed every two hours, for 6 days. The mean FAM level in the HF group was 5038.0 ± 1529.1 ng/g, while for LF, 2178.7 ± 824.9 ng/g (p < 0.05). The mean HF fecal production was 182.6 ± 36.2 g DM/day and 117.5 ± 12.6 g DM/day for LF (p < 0.05). There were no differences in terms of mean defecation rate, passage, and retention times between groups. The results suggest that dietary fiber affects the FAM detection, and this should be taken into consideration before conducting experiments using fecal samples as a source of reproductive hormones profiling., (Copyright © 2022 Elsevier Inc. All rights reserved.)