OBJECTIVE: This study compares the new Motor Unit Number Estimation (MUNE) technique, MUNIX, with the more common incremental stimulation MUNE (IS-MUNE) with respect to reproducibility in healthy subjects and as potential biomarker of disease progression in patients with ALS. METHODS: Thirteen ALS patients and 48 control subjects were prospectively investigated - both groups were studied with MUNIX and IS-MUNE applied on the abductor digiti minimi (ADM) muscle. Additional retest was performed on 14 control subjects. Follow-up tests were carried out on 6 patients. The analysis included measures of reproducibility (Intraclass Correlation Coefficient (ICC)) and diagnostic performance (Receiver Operating Characteristic (ROC) analysis). RESULTS: Test-retest reproducibility was low to moderate for MUNIX and IS-MUNE (ICC=0.38 and 0.56, respectively). Repeated MUNIX and IS-MUNE measurements on the same subject had a mean percentage difference (MPD) of 20% and 46%, respectively (p=0.039). In the control group, the coefficient of variation was markedly lower for MUNIX than for IS-MUNE (26% and 44%, respectively, p