OBJECTIVE The mechanisms underpinning the association between chronic stress and gut health are poorly understood. We aimed to investigate the relationship between bacterial produced short-chain fatty acids, gut barrier function, and stress measures. METHODS A fecal sample, hair sample, and questionnaire data were collected from 113 Belgian children (8-16 years old). Biological measures of stress included hair cortisol (most proximal 3 cm) and 5-minute heart rate variability (high frequency). Self-report measures of stress included emotional problems and negative events. Fecal calprotectin was determined as a marker of intestinal inflammation and an indirect indicator of gut barrier integrity. Fecal short-chain fatty acids (butyrate, propionate, acetate, valerate, isobutyrate, and isovalerate) were measured with gas chromatography. Linear regression analyses were adjusted for sex, age, socioeconomic status, body mass index, fiber intake, and protein intake. RESULTS Emotional problems were significantly associated with higher butyrate (β = 0.263), valerate (β = 0.230), isovalerate (β = 0.231), and isobutyrate (β = 0.233). Heart rate variability reflecting higher parasympathetic activity was related to lower valerate levels (β = -0.217). Hair cortisol was not associated with the short-chain fatty acids. None of the stress measures and none of the fecal short-chain fatty acids were significantly related to fecal calprotectin. CONCLUSIONS In healthy children, the impact of chronic stress is manifested more obviously in short-chain fatty acids than in intestinal inflammation as measured by levels of calprotectin. Despite the rather counterintuitive associations with butyrate, these results point to the need for further research on gut microbiome composition.