In this study, peripheral microcirculatory response to arterial and venous cannulation was studied. Eleven Sprague-Dawley rats were evaluated in two groups. Group I was the control group and received no cannulation (N = 6). After cremaster muscle isolation, the following parameters were evaluated at the microcirculatory level: vessel diameters, red blood cell (RBC) velocities, capillary density, and leukocyte and lymphocyte behavior (rollers, stickers, transmigrating white blood cell [WBC]). In group II, vessel cannulation was performed (N = 5). Before cremaster muscle isolation, the right femoral artery was cannulated for mean arterial blood pressure measurements; the right carotid artery was cannulated for arterial partial oxygen pressure (PaO2), arterial partial carbon dioxide pressure (PaCO2), and pH measurements; and the left jugular vein was cannulated for central venous pressure evaluation. Microcirculatory measurements as in group I were also taken. In the cannulated animals, arterial RBC velocity was 18.1% faster, whereas venous RBC velocity was 15.7% decreased (p< 0.05). In addition, leukocyte activation Increased and was confirmed by a 254.6% rise in rolling leukocytes, a 59.7% increase in rolling lymphocytes, and a 67.2% increase in leukocyte “stickers‘’ (p< 0.05). In group II, functional capillary perfusion was decreased by 44.9%, and 4.8% higher endothelial edema indexes were found (p< 0.05). In conclusion, this study has proven that, despite its clinical importance during vital signs monitoring, cannulation procedures may significantly Impair peripheral microcirculatory hemodynamics. For this reason, cannulation procedures should be designed with caution and should be reported in experimental methods.