Objective and design: Hypertonic saline administered during fluid resuscitation may mitigate endothelial glycocalyx (EG) shedding and inflammation. The objective of this pilot randomised controlled trial was to measure the effect of hypertonic saline, compared to isotonic saline, on biomarkers of EG shedding and inflammation in emergency department patients with suspected sepsis. Methods: Patients received either 5 mL/kg of 3% saline (hypertonic group, n = 34) or 10 mL/kg of 0.9% saline (isotonic group, n = 31). Change in serum biomarker concentrations of syndecan-1, hyaluronan, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, interleukin-6, -8, -10, interferon-γ, neutrophil gelatinase-associated lipocalin and resistin were compared from baseline (T0) to after fluid (T1), 3 h (T3) and 12–24 h (T24) later, as was serum osmolality, using linear mixed effects models. Results: The hypertonic group had significantly increased mean serum osmolality compared to the isotonic group at T1 (P <.001) and T3 (P =.004). Minor differences were found in some biomarker outcomes, including a decreased fold-change in syndecan-1 at T1 (P =.012) and in interleukin-10 at T24 (P =.006) in the isotonic group, compared to the hypertonic group. Conclusions: Although a single bolus of hypertonic saline increased serum osmolality, it did not reduce biomarkers of EG shedding or inflammation, compared to patients that received isotonic saline. Trial registration: ANZCTR.org.au, ACTRN12611001021965, Registered on 23rd September 2011.