As of now the relationship between glycocalyx degradation and microcirculatory perfusion abnormalities in non-septic critical ill patients is unclear. In addition, conjunctival sidestream dark field-imaging for the purpose of glycocalyx thickness estimation has never been performed. We aimed to investigate whether changes in glycocalyx thickness in non-septic patients are associated with microcirculatory alterations in conjunctival and sublingual mucosa. In this single-centre prospective observational study, using techniques for direct in-vivo observation of the microcirculation, we performed a single measurement of microcirculatory perfusion parameters and visualized glycocalyx thickness in both ocular conjunctiva and sublingual mucosa in mixed cardio surgical (n = 18) and neurocritical patients (n = 27) and compared these data with age-matched healthy controls (n = 20). In addition we measured systemic syndecan-1 levels. In the sublingual and conjunctival region we observed a significant increase of the perfused boundary region (PBR) in both neuro-critical and cardiac surgical ICU patients, compared to controls. There was a significant increase of syndecan-1 in ICU patients comparing with controls and in cardiac patients comparing with neurological (120.0[71.0-189.6] vs. 18.0[7.2-40.7], p < 0.05). We detected a weak correlation between syndecan-1 and sublingual PBR but no correlations between global glycocalyx damage and conjuctival glycocalyx thickness. We found significantly lower perfused vessel density (PVD) of small vessels in sublingual mucosa in patients after cardiac surgery in comparison with healthy subjects. In neuro-critical, but not cardiac surgery patients conjunctival TVD and PVD of small vessels were found to be significantly lower in comparison with controls.