Background: The endothelial glycocalyx controls vascular permeability, cellular signaling, blood-endothelial cell adhesion, extravasation, and transmission of shear stress signals. Burn injury compromises integrity of this layer increasing vascular permeability, which is further exacerbated by large volumes of (intravenous) crystalloids. We have shown that enteral resuscitation is able to reverse burn-induced acute kidney injury (AKI), and herein, we present a follow-up examination of the integrity of the glycocalyx layer and its relationship with renal dysfunction after burn injury. Materials and methods: Anesthetized Yorkshire pigs sustained 40% total body surface area full-thickness contact burns and recovered in metabolic cages for one of three treatments: no fluids (oral or intravenous); (n = 6), ad libitum water (n = 6), or volume-matched oral rehydration solution (ORS; n = 6) for 48 h. Urine and blood were collected at baseline (BL), 6, 12, 24, 32, and 48 h after burn at which point kidneys were harvested. Results: In no fluid and water groups (but not ORS), plasma levels of glycosaminoglycans (GAGs) were elevated after burn (P ≤ 0.031). Syndecan-1 was elevated by 6 h after burn in all animals, but levels declined by 24 h with enteral fluids. Urinary GAGs in the no-fluid group were elevated after burn. No differences among treatments were detected in syndecan-1 levels, or glomerular lectin within the kidney. Conclusions: Collectively, these data demonstrate that ORS prevented increases in circulating GAGs. Furthermore, an inexpensive and simple method for detecting GAGs provides a sensitive measure of endotheliopathy after burn.