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The microvascular endothelium and endothelial cells are primary targets during graft rejection. This study measured endothelial cell damage by assessing microvascular permeability, and evaluated the rate of surgical trauma during the acute phase of transplant rejection. The authors developed an experimental model that allows for simultaneous measurement of leukocyte activation and albumin leakage to assess endothelial barrier function exposed to transplantation-induced trauma. Eighteen composite tissue isograft transplantations were performed between genetically identical rats, and 18 cremaster muscle flaps were subjected to the same ischemic insult as the transplantation group to allow for a control group with which to compare the effects of ischemia without concomitant transplantation trauma. At 24 hours, 72 hours, and 7 days follow-up, microvascular permeability, leukocyte activation, functional capillary perfusion, and endothelial edema index were measured in both groups. There was a significant increase in the permeability index of the transplantation group at 24 hours follow-up (p = 0.005), with an increasing trend at 72 hours and 7 days follow-up (p < 0.001). The permeability index; number of rolling, sticking, and transmigrating leukocytes; and rolling and sticking lymphocytes were significantly greater in the transplantation group at all follow-up points compared with the ischemic control group (p < 0.001). These findings demonstrate the added effect of transplantation trauma to ischemia and reperfusion injury. We observed increased leukocyte—endothelial interactions and acute destruction of endothelial cell barrier function during the acute rejection period after composite limb tissue transplantation.