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Abstract Endothelial glycocalyx (EG) shedding is associated with septic shock and described following intravenous (IV) fluid administration. To investigate the possible impact of IV fluids on the pathobiology of septic shock we investigated associations between biomarkers of EG shedding and endothelial cell activation, and relationships with IV fluid volume. Serum samples were obtained on admission (T0) and at 24 h (T24) in patients undergoing haemodynamic resuscitation for suspected septic shock in the emergency department. Biomarkers of EG shedding—Syndecan-1 (Syn-1), Syndecan-4 (Syn-4), Hyaluronan, endothelial activation—Endothelin-1 (ET-1), Angiopoeitin-2 (Ang-2), Vascular Endothelial Growth Factor Receptor-1(VEGF-1) and leucocyte activation/inflammation—Resistin, Neutrophil Gelatinase Associated Lipocalin (NGAL) and a marker of cardiac stretch—Pro-Atrial Natriuretic Peptide (Pro-ANP) were compared to the total IV fluid volume administered using Tobit regression. Data on 86 patients (52 male) with a mean age of 60 (SD 18) years were included. The mean fluid volume administered to T24 was 4038 ml (SD 2507 ml). No significant association between fluid volume and Pro-ANP or any of the biomarkers were observed. Syn-1 and Syn-4 were significantly correlated with each other (Spearman Rho 0.43, p