The motor unit number index (MUNIX) refers to an electrophysiologic technique that measures the approximate number of motor units using the surface electromyographic interference pattern (SIP) recorded during voluntary contraction. This study was done to assess the reproducibility of MUNIX performed on hypothenar muscles in 62 normal controls and 22 amyotrophic lateral sclerosis (ALS) patients. Inter- and intraoperator correlation coefficients for MUNIX were 0.74 and 0.86, respectively, in normal controls, and 0.95 and 0.93, respectively, in ALS patients (P < 0.01 in all). Inter- and intraoperator coefficients of variation for MUNIX measurements were 17.5% and 15.3%, respectively, in normal controls, and 23.7% and 24.0%, respectively, in ALS patients. This study shows a good correlation for MUNIX between intra- and interoperator results in both normal controls and ALS patients. The test-retest variability seems to be greater in ALS patients compared with normal controls, but this will need to be confirmed in future studies. Sources of variability should be identified and corrected for clinical use.