Introduction: The endothelial glycocalyx is increasingly considered as an intravascular compartment that protects the vessel wall against pathogenic insults. Objective: The purpose of this study was to translate an established experimental method of estimating capillary glycocalyx dimension into a clinically useful tool and to assess its reproducibility in humans. Methods: We first evaluated by intravital microscopy the relation between the distance between the endothelium and erythrocytes, as measure of glycocalyx thickness, and the transient widening of the erythrocyte column upon glycocalyx compression by passing leukocytes in hamster cremaster muscle capillaries. We subsequently assessed sublingual microvascular glycocalyx thickness in 24 healthy males using orthogonal polarization spectral imaging. In parallel, systemic glycocalyx volume (using a previously published tracer dilution technique) as well as cardiovascular risk profiles were assessed. Results: Estimates of microvascular glycocalyx dimension from the transient erythrocyte widening correlated well with the size of the erythrocyte-endothelium gap (r=0.63). Measurements in humans were reproducible (0.58±0.16 and 0.53±0.15 µm, coefficient of variance 15±5%). In univariate analysis, microvascular glycocalyx thickness significantly correlated with systemic glycocalyx volume (r=0.45), fasting plasma glucose (r=0.43) and HDL-cholesterol (r=0.40) and correlated negatively with LDL-cholesterol (r= -0.41) as well as body mass index (r= -0.45) (all p<0.05). Conclusion: The dimension of the endothelial glycocalyx can be measured reproducibly in humans and is related to cardiovascular risk factors. It remains to be tested whether glycocalyx dimension can be used as an early marker of vascular damage and whether therapies aimed at glycocalyx repair can protect the vasculature against pathogenic challenges.