Thenar motor unit number estimate (MUNE) reproducibility was assessed in 20 patients with amyotrophic lateral sclerosis (ALS) and 16 normal subjects using the multiple point stimulation (MPS) technique. The MUNE was calculated by dividing the thenar compound muscle action potential negative-peak (n-p) area by the mean n-p area of 10 lowest threshold, all-or-nothing, surface-recorded motor unit action potentials. Two trials (test-retest) were performed by the same examiner either on separate days or on the same day with new electrode placements. The mean test MUNE was 43.4 (SD: 35.9, range: 6-145) for ALS patients and 219.4 (SD: 80.8, range: 122-368) for normal subjects. Test-retest MUNE differences were not significant for ALS patients or normal subjects. The test-retest correlation coefficient (r) was 0.99 for ALS patients and 0.85 for normal subjects. The mean difference between test-retest values was 10% for ALS patients and 17% for normal subjects. Test-retest reproducibility of the thenar MUNE using the MPS technique is high in both ALS patients and normal subjects. The reliability of the MPS technique in estimating motor unit numbers may make it a useful outcome measure in following the course of patients with progressive lower motor neuron disease, especially those enrolled in experimental drug trials.