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Perturbation of the endothelial glycocalyx has been suggested to be an early event in the development of endothelial dysfunction during increased cardiovascular risk exposure. Previously, we showed in anesthetized rats that acute enzymatic degradation of the endothelial glycocalyx was associated with impaired insulin-mediated glucose disposal, suggesting a role of the glycocalyx in regulation of glucose homeostasis. In the current study, we tested the acute effect of enzymatic glycocalyx treatment on glucose tolerance and insulin sensitivity in conscious rats. In rats that had had their femoral artery and vein cannulated 1 week before the experiment, intravenous glucose and insulin tolerance tests were performed before and after acute degradation of the endothelial glycocalyx using an intravenous bolus of hyaluronidase. During the intravenous glucose tolerance test, glucose tolerance did not differ between the hyaluronidase-treated and control animals, but was associated with a 1.5-fold higher plasma insulin level in the former group. Further, glycocalyx treatment significantly decreased insulin-mediated glucose disposal during intravenous insulin tolerance tests, as observed from the reduced insulin-mediated glucose disposal rate from 3.3±0.1%/min in control rats to 2.6±0.2%/min in the hyaluronidase-treated rats. These results support a role for the endothelial glycocalyx in the regulation of peripheral insulin sensitivity. The reduction in insulin sensitivity during acute glycocalyx damage, however, does not have an immediate effect on glucose tolerance because of a compensatory increase in plasma insulin levels.