Systemic parameters, microvascular and capillary hemodynamics were studied in the hamster window chamber model before and after hyaluronan degradation by intravenous injection of Streptomyces hyaluronidase (100 U, 40-50 U/ml plasma). Glycocalyx permeation was estimated using fluorescent markers of different molecular size (40, 70 and 2000 kDa), and electrical charge. Systemic parameters (blood pressure, heart rate, blood gases) and microhemodynamics (vascular tone, velocity and blood flow) remained statistically unchanged after injection of hyaluronidase, when compared to inactivated hyaluronidase. Conversely, capillary hemodynamics were drastically affected. Functional capillary density (FCD), the capillaries perfused with red blood cells (RBCs), decreased by 35%, capillary hematocrit (Hct) of the remaining functional capillaries increased from 16% to 27%, and penetration of 70 kDa fluorescent marker increased. Furthermore, plasma-only perfused capillaries statistically increased 30 min after hyaluronidase. The decrease in FCD accounted for the increase in RBC flux in the remainder of the capillaries, since the same number of RBCs had to traverse a reduced number of capillaries. Flux balances showed a reduction from baseline of 11% for the RBC flux and 20% for the plasma flux after treatment. These discrepancies are within the margin of error of the techniques used, and could be explained by accounting for RBC over-velocity compared to plasma. These findings suggest that the decrease in the glycocalyx leads to capillary perfusion impairments.