Diarrhea is the leading cause of mortality in neonatal dairy calves in US during the first 4 weeks of life. Therefore, studying physiological adaptations of the gastrointestinal (GI) tract epithelial cells to diarrhea is of great importance to the dairy industry. This study aimed to isolate RNA from exfoliated cells present in fresh fecal samples from neonatal dairy calves and evaluate the GI tract physiological changes in response to mild diarrhea via transcriptomic profiling. Newborn Jersey calves (n = 8) were housed in individual pens from birth to 5 wk of age at Oregon State University Dairy Unit. Calves had ad-libitum access to water and starter grain and were fed 2× daily a total of 5.6 L whole milk. Blood samples were collected weekly for metabolic and inflammatory profiling during the experiment. Fecal score (FS), body weight (BW) and starter intake were recorded throughout the experiment. RNA isolated from fresh fecal samples collected weekly was used for RT-qPCR analysis. Data were analyzed using the PROC MIXED procedure of SAS. Orthogonal contrasts were used when a time effect (P < 0.05) was observed to evaluate linear or quadratic effects over time. Starter intake and BW increased (P < 0.01) over time. FS was greatest (2.6 ± 0.3; P < 0.01) at 2 wk. Blood glucose and BHBA had a linear (P ≤ 0.04) effect over time. Biomarkers of liver function, cholesterol, albumin, and paraoxonase had a linear response (P < 0.01) over time. In blood, reactive oxygen metabolites reached a peak at wk 2, which was explained by a positive quadratic effect (P < 0.01). Similarly to FS, blood inflammatory biomarkers, IL-6, ceruloplasmin, and haptoglobin had a positive quadratic effect (P ≤ 0.04). The inflammation-related genes TLR4, TNFA, IL8 and IL1B in fecal RNA had a positive quadratic effect (P = 0.05). A negative quadratic effect (P ≤ 0.05) was observed on cell membrane transporters genes, AQP3 and SLC5A1. Overall, our findings support the use of fecal RNA as a non-invasive tool to evaluate transcriptomic alterations during diarrhea in neonatal dairy calves. Future research could use such method to observe physiological alterations in response to dietary treatments.