The gastrointestinal inflammatory response may play a role in the susceptibility of preterm infants for infections. We previously reported a trend toward lower endogenous infection morbidity after enteral supplementation of neutral and acidic oligosaccharides (SCGOS/LCFOS/AOS). We hypothesize that enteral supplementation of prebiotics may decrease infectious morbidity by reducing intestinal inflammation. Therefore, we aimed to determine the effect of enteral supplementation of prebiotics on intestinal inflammation, as measured by fecal IL-8 (f-IL-8) and calprotectin (f-calprotectin), in preterm infants. In a randomized controlled trial, infants with a GA <32 wk and/or birth weight <1,500 g received enteral supplementation of prebiotics or placebo (maltodextrin) between d 3 and 30 of life. F-IL-8 and f-calprotectin was assessed at baseline, d 7, 14, and 30 of life. In total, 113 infants were included. Baseline patient and nutritional characteristics were not different in the SCGOS/LCFOS/AOS (n = 55) and the placebo group (n = 58). Enteral supplementation of prebiotics had no effect on f-IL-8 and f-calprotectin. F-IL-8 and f-calprotectin were strongly correlated at all time points (p < 0.001). In conclusion, enteral supplementation of prebiotics (SCGOS/LCFOS/AOS) does not affect f-IL-8 and f-calprotectin levels in preterm infants.Abbreviations: GEE, generalized estimating equations; SCGOS/LCFOS/AOS, a mixture of neutral and acidic oligosaccharides; galacto-oligosaccharides/fructo-oligosaccharides/acidic oligosaccharides; f-calprotectin, fecal calprotectin; f-IL-8, fecal IL-8; NEC, necrotizing entero colitis