The purpose of this study was to evaluate stress by comparing blood glucose and serum corticosterone levels after repeated blood sampling over 2 h (five time points) in anesthetized noncannulated rats to those of nonanesthetized jugular-cannulated animals. Noncannulated rats underwent isofluorane anesthesia for the duration of the blood sampling at each time point. For both sampling methods, blood glucose concentrations increased after the initiation of blood sampling, peaked at significantly increased (noncannulated rats, P < 0.01; cannulated rats, P < 0.01) concentrations at 0.5 h, and decreased thereafter until the end of the assessment period. Despite the observed fluctuations, blood glucose concentrations remained within normal ranges. Similarly, corticosterone concentrations increased significantly (noncannulated rats, P < 0.01; cannulated rats, P < 0.001) to peak values at 0.5 h. However, corticosterone was significantly lower at the 1- (P < 0.01) and 2-h (P < 0.05) time points in cannulated rats compared with anesthetized rats. Therefore, although both sampling methods are similar regarding blood glucose and corticosterone peak concentrations and time-to-peak, stress was slightly less in the cannulated rats than the rats that underwent repeated anesthesia.