The luminal surface of blood vessels is coated with a size limiting, specialised, composite endothelial glycocalyx-surface layer (EG-SL) which has a fundamental role in microvessel permeability. EG-SL disruption occurs in major global diseases (inc. diabetes, atherosclerosis, sepsis, advanced kidney disease). Advancing knowledge about the EG-SL is seriously limited by difficulties in detecting, imaging and accurately quantifying the EG-SL, particularly in functioning vessels in vivo. We have developed a new technique for studying the EG-SL in vivo, and used this system to demonstrate changes in the EG-SL in an animal model of early kidney disease. Male Sprague Dawley rats were injected with either Puromycin Aminonucleoside to cause Nephrosis (PAN) or PBS (sham controls). Mesenteric microvessels of pentobarbitone-anaesthetised (60 µg per g in weight) rats were exposed, cannulated, and perfused with octadecyl-rhodamine-B chloride (R18: membrane label) and Fluorescein-Isothiocyanate-conjugated Wheat Germ Agglutinin (FITC-WGA lectin: EG-SL label), and imaged using Confocal Microscopy (Nikon D-eclipse C1, CM) with a 60x objective (1.4 NA, Nikon). Vessels of sham injected rats were then perfused with neuraminidase for 20min, an enzyme that cleaves sialic acid residues within the glycocalyx, and re-imaged. EG-SL depth was estimated based on the anatomical distance between peak fluorescence of FITC-WGA and R18 signals (FIJI/IMAGEJ, NIH) at the maximum vessel width. EG-SL depth in sham rats was 288±24nm (n=5). This value was significantly reduced to 185±32nm following perfusion of the same vessel with neuraminidase (p<0.01, paired t-test). EG-SL depth in PAN was 147±23nm (n=5), demonstrating significantly reduced EG-SL depth in these proteinuric rats compared with sham-treated rats (p<0.01, unpaired t-test). We have demonstrated a sensitive technique for measuring changes to EG-SL depth in vivo using CM. EG-SL depth is significantly reduced in proteinuric PAN-treated rats, perhaps a contributing factor to the increased systemic permeability observed in proteinuric patients.