Purpose: Endocan, a component of the endothelial glycocalyx (EG), has been linked with respiratory failure in sepsis. This study explored the temporal patterns of three EG biomarkers, including endocan, and their relationships with inflammation and respiratory failure. Materials and methods: Plasma endocan, syndecan-1, and hyaluronan concentrations were measured in Emergency Department (ED) patients with sepsis due to pneumonia (n = 44) on ED arrival (T0), 1 h (T1), 3 h (T3) and 12–24 h (T24) later, with change over time tested using mixed regression models. Biomarker associations with inflammatory cytokine concentrations and with respiratory failure on days 1, 2 or 3, need for mechanical ventilation and 30-day mortality were also tested. Results: Endocan concentration significantly decreased over time (T0–T24, P = 0.003) whereas both syndecan-1 (T0–T3, P = 0.010; T0–T24, P < 0.001) and hyaluronan (T0–T1, P = 0.010; T0–T3, P < 0.001; T0–T24, P = 0.003) significantly increased over time. Increased syndecan-1 was significantly correlated with neutrophil activation biomarkers and significantly increased the odds of respiratory failure (OR 1.18, 95% CI 1.05–1.33, P = 0.004), need for mechanical ventilation (OR 1.24, 95% CI 1.04–1.48, P = 0.014) and 30-day mortality (OR 1.29, 95% CI 1.07–1.55, P = 0.008). Conclusion: Syndecan-1, but not endocan, was associated with neutrophil activation and was the best EG biomarker predictor of adverse clinical outcomes.