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Although the beneficial effects of fiber supplementation on overall health and the gut microbiome are well-known, it is not clear whether fiber supplementation can also alter the concentrations of lipopolysaccharide-binding protein (LBP), a marker of intestinal permeability. A secondary analysis of a previously conducted study was performed. In the randomized-order, placebo-controlled, double-blinded, cross-over study 20 healthy, young participants consuming a low-fiber diet at baseline were administered a daily dose of 12 g of prebiotic fiber compared with a placebo over a period of 4 weeks with a 4-week washout between arms. In this secondary analysis, we hypothesized that the fiber supplement would reduce LBP concentration. We further hypothesized that lecithin cholesterol acyltransferase activity, a measure of high-density lipoprotein functional capacity, would be altered. Fiber supplementation did not significantly alter LBP concentration or lecithin cholesterol acyltransferase activity in the overall cohort. However, in a subgroup of individuals with elevated baseline LBP concentrations, fiber supplementation significantly reduced LBP from 9.27 ± 3.52 to 7.02 ± 2.32 µg/mL (P = .003). Exploratory analyses found positive correlations between microbial genes involved in lipopolysaccharide synthesis and conversely negative correlations with genes involved in antibiotic synthesis and LBP. Positive correlations between LBP and multiple sulfated molecules including sulfated bile acids and perfluorooctanesulfonate, and ibuprofen metabolites were also found. These findings highlight multiple environmental and lifestyle factors such as exposure to industrial chemicals and medication intake, in addition to diet, which may influence the association between the gut microbiome and gut barrier function., Competing Interests: Author Declarations The authors have no conflict of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)