Introduction Amyotrophic Lateral Sclerosis (ALS) is characterized by progressive loss of motor units, but electromyography (EMG) with quantitative motor unit potential (MUP) analysis does not directly quantify the denervation process. A more extensive clinical use of Motor Unit Estimation Methods: (MUNE), like MScan MUNE, could hence be of potentially great value. Muscle Velocity Recovery Cycle (MVRC) recordings can provide in vivo information about changes in muscle fiber membrane properties, and may enlighten the effect of the denervation process on the muscle fibers. The aim of this study was to examine the clinical utility of MScan MUNE and MVRC in ALS patients; which also contribute to a better understanding of disease pathophysiology. Methods 27 ALS patients and 23 age and sex-matched healthy control subjects were included. All participants were evaluated electrophysiologically by a standard quantitative EMG and by the two novel methods: MScan MUNE and MVRC, all in the anterior tibial muscle (TA). MScan MUNE, MVRC and EMG parameters were compared between ALS patients and healthy controls using parametric unpaired t-tests. ROC curves were used to test how these methods distinguish patients from healthy controls. MVRC parameters were correlated to EMG results and MScan parameters using linear regression analysis. Results The MScan MUNE value for ALS patients (55 (95% CI: 42–68)) was significantly lower than for the healthy controls (108 (95% CI: 98–117)) (p 5 sites) and controls. And there was no significant correlation between MVRC and MUP parameters or incidence of spontaneous activity. Conclusion The novel MScan MUNE method is a sensitive quantitative measure of loss of motor units in ALS patients in TA. The finding of no difference in MVRC for ALS patients compared to healthy controls indicate, that the muscle fiber membranes are not prominently affected by the denervation process. This may be explained by the compensatory process of collateral reinnervation, which initially is strong and ensures that the vast majority of muscle fibers are innervated and behave normally.