© 2022 Taylor & Francis Group, LLC.Background: Although in vitro methods have disadvantages, they are still commonly used to measure nerve conduction velocity (NCV) in experimental studies. Therefore, this study was designed to demonstrate the effect of the surgical procedures required for in vitro methods on nerve fibers and the effect of in vivo and in vitro methods on the results of electrophysiological measurements. Methods: Rats were assigned to the in vivo (control-1, injury-1, and diabetic-1) and in vitro (control-2, injury-2, and diabetic-2) groups. The NCV and compound action potential amplitudes were measured, and the nerve fibers were histologically examined. Results: Damaged axons and myelin sheaths were observed in the control-2 group. The electrophysiological values of the in vitro groups were lower than those of the in vivo groups. Furthermore, these values were lower for the diabetic and injury groups than for the control groups. Conclusions: This study showed that the surgical procedures required for the in vitro method reduced the measured values. Owing to the previous and current disadvantages of the in vitro method, the in vivo method was more sensitive for the NCV measurement. Moreover, measurements can be performed using the current in vivo method for small nerve fibers.