The aim of this study was to analyse MScanFit MUNE, D50 and step% values elicited from the CMAP scan curves of thenar and hypothenar muscles in patients with amyotrophic lateral sclerosis (ALS) and healthy controls. Thirty-four ALS patients (31 definite, 1 probable and 2 possible, according to Awaji criteria) and 24 healthy controls were included. CMAP scan curves were recorded from both thenar and hypothenar muscles by applying 500 stimuli. Step% and D50 values were calculated semi-automatically and MScanFit MUNE was calculated by using the freeware version of Qtrac software. Mean MScanFit MUNE and D50 values were lower and step% values were higher significantly in patients (48.8, 25.0, 32.5 in thenar muscles and 66.5, 28.9, 17.0 in hypothenar muscles, respectively) as compared to healthy controls (112.5, 45.6, 4.0 in thenar muscles and 124.5, 46.6, 1.1 in hypothenar muscles; p < 0.001). Step% provided the best discrimination by ROC analysis (area under the curve was 0.904 for thenar and 0.923 for hypothenar muscles). MScanFit MUNE and step% were significantly more abnormal in the thenar as compared to hypothenar muscles. MScanFit MUNE values were correlated significantly with step% and D50 values in both muscle groups (Spearman’s rho, p < 0.001). Step% and MScanFit MUNE are parameters correlated with each other and they are sensitive tools in revealing the severities and the courses of the electrophysiological abnormalities in ALS.