The analysis of glucocorticoids excreted in feces is becoming a widespread technique for determining animal wellbeing in a wide variety of settings. In the present study an extraction protocol and an ELISA assay for quantifying fecal corticosterone metabolites (FCM) in BALB/c and C57bl/6 mice were validated. Lower ratios of solvent (ethanol) to mass of fecal sample were found to be sufficient in extracting FCM compared to what has been reported previously. Feeding mice a high energy diet, high in fat content (60% of calories from fat), significantly lowered the FCM excretion, approximately halving the FCM output. This diet also reduced the fecal mass voided to approximately a third of that of the regular diet. The two reductions were not correlated. A difference in defecation pattern was seen between the two strains, with the BALB/c mice having a more pronounced diurnal rhythm compared to the C57bl/6 mice. Furthermore, throughout the experiment, the C57bl/6 mice excreted significantly higher levels of FCM compared to the BALB/c mice. The mice were also challenged with synthetic adrenocorticotropic hormone (ACTH) and dexamethasone (DEX). The effect of the challenges could readily be detected, but had a considerably lesser impact on data than did the difference in diet. The study demonstrates some problematic consequences of expressing FCM excretion as a measure of fecal dry mass. The study also serves to emphasize the caution that must be exercised when interpreting FCM excretion in conjunction with an uncontrolled or varied diet, or perturbations of gastro-intestinal functioning.