The nociceptive withdrawal reflex (NWR) is a polysynaptic spinal reflex protecting the body from harmful stimuli. Two different methods to assess its' threshold (NWR-T) have been part of clinical trials concerning the evaluation of the nociceptive system in the human body. NWR-T's are gathered by stimulation at the sole of the foot and over the sural pathway. Consequently, EMG analyzes the muscle activity over the biceps femoris and tibialis anterior muscle. Past studies favor stimulation at the sole of the foot. The two methods were compared concerning retest-reliability and subjective pain ratings. The retest-reliability was tested over a period of 21 days using an up-down staircase method. Reliability was evaluated with a Bland Altman agreement analysis. Subjective pain ratings were evaluated with a numeric rating scale (NRS). NWR-T assessment was successful for all subjects. The EMG muscle activity had larger reflex amplitudes for measurements of the tibialis anterior muscle. NWR-T values showed greater variability than NRS values. The retest-reliability over a period of 21 days showed stable NWR-T results for both stimulation sites, but superior reliability was gathered with stimulation at the sole of the foot. Subjects rated stimulation over the sural pathway as more painful. The NWR-T upholds reliable measurements over a longer period of time and seems to be a stable measure for pain condition. Reliability estimations, EMG recordings, and subject's rating show stimulation at the sole of the foot could be the better choice.