In amyotrophic lateral sclerosis (ALS), progressive motor neuron loss causes severe weakness. Functional measurements tend to underestimate the underlying pathology because of collateral reinnervation. A more direct marker of lower motor neuron loss is of significant importance. We evaluated high-density motor unit number estimation (MUNE), as compared with the ALS Functional Rating Scale (ALSFRS) and maximal compound muscle action potential (CMAP) amplitude, for monitoring and classifying disease progression. MUNE showed good reproducibility (intraclass correlation coefficient = 0.86). MUNE showed a significantly greater decrease than the ALSFRS, the Medical Research Council (MRC) scale, and CMAP amplitude. Patients could be stratified into groups with rapidly or slowly progressive disease based on a decrement in MUNE at 4 months from baseline; ALSFRS score at 8 months was significantly lower in the rapidly progressive group. MUNE was sensitive to motor neuron loss early in the disease course when compared to other clinical measures. Stratification of patients based on a decrease in MUNE seems feasible.