© 2016 American Society for Nutrition.Background: Very preterm infants often receive donor milk from mothers who deliver at term, but its composition differs from that of their own mother's milk. Because breast-milk glucocorticoids can support developing neonates, we explored concentration variability within and between mothers. Objective: We hypothesized that breast-milk glucocorticoid concentrations would be higher after very preterm delivery [gestational age (GA) < 32 wk; study 1] and would follow the diurnal rhythm of maternal adrenocortical activity (study 2). Methods: Study 1 assessed differences inmilk cortisol, cortisone, and the cortisone-to-(cortisol+cortisone) ratio of mothers who delivered at (median) GA: 28.6 wk or at term weekly during the first month postpartum. Study 2 assessed variations in milk cortisol, cortisone, and the cortisone-to-(cortisol+cortisone) ratio over 24 h, and tested Pearson correlations between milk and salivary concentrations in mothers who delivered at term (median GA: 38.9 wk) during week 4 postpartum. In these studies, foremilk glucocorticoidsweremeasured by liquid chromatography-tandemmass spectrometry. Associations ofmilk cortisol,milk cortisone, and the milk cortisone-to-(cortisol+cortisone) ratio with prematurity (study 1) or collection time (study 2) were studied with longitudinal data analyses. Results: In study 1, giving birth to a very preterm infant was associated with reductions in milk cortisol and cortisone concentrations of 50%(b: 0.50; 95%CI: 0.26, 0.99; P = 0.05) and 53%(b: 0.53; 95%CI: 0.30, 0.93; P = 0.03), respectively, when adjusted for collection time. In study 2, concentrations of milk cortisol and cortisone were associated with collection time (bothP < 0.01), peaking at;0700.Milk and salivary concentrations of cortisol (r =0.92, P < 0.01) and cortisone (r= 0.93, P < 0.01) as well as the cortisone-to-(cortisol+cortisone) ratio (r = 0.64, P < 0.01) were correlated with one another. Conclusions: Breast-milk glucocorticoid concentrations follow the diurnal rhythm of maternal hypothalamus-pituitaryadrenal axis activity and are lower in mothers who deliver very preterm.