Objective To evaluate how the motor unit number index (MUNIX) is related to high-density motor unit number estimation (HD-MUNE) in healthy controls and patients with amyotrophic lateral sclerosis (ALS). Methods Both MUNIX and HD-MUNE were performed on the thenar muscles in 18 ALS patients and 24 healthy controls. Patients were measured at baseline, within 2 weeks, and after 4 and 8 months. Clinical evaluation included Medical Research Council (MRC) scale and the ALS functional rating scale (ALSFRS). Results There was a significant positive correlation between MUNE and MUNIX values in ALS patients (r = 0.49 at baseline; r = 0.56 at 4 months; r = 0.56 at 8 months, all p < 0.05), but not in healthy controls. After 8 months, both MUNE and MUNIX values of the ALS patients decreased significantly more compared to MRC scale, ALS functional rating scale (ALSFRS) and compound muscle action potential (CMAP) (p < 0.05). There was no significant difference in relative decline of MUNIX and HD-MUNE values. Conclusions In ALS patients, MUNIX and HD-MUNE are significantly correlated. MUNIX has an almost equivalent potential in detecting motor neuron loss compared to HD-MUNE.