Background: What is the number and size of motor units, and axonal excitability profile in the early stages of muscle weakness in ALS compared to controls? Methods: We enrolled ALS patients with APB manual strength testing rated four or four-minus (ALS:4-arm) and four-plus (ALS:4+ arm) and control participants >35 years-old from the University of Toronto, University of Alberta and Universidade Federal de Sao Paulo. Mean±SD, one-way ANOVA and ANCOVA of ALSFRS-R, PUMN Score, MUNIX, MUSIX, and nerve-excitability testing using QTRAC TROND protocol were reported. Results: Twenty-five ALS patients and 63 controls were included. Mean MUNIX was significantly lower (p<0.0001) and MUSIX was significantly higher (p<0.001) in both ALS groups compared to controls. Mean strength-duration time constant in the ALS:4- arm (0.50ms±0.11; p<0.05) and superexcitability in both ALS groups (ALS:4- -29.05%±9.24, ALS:4+ -27.67%±8.03; p<0.05) were relatively increased, supporting axonal hyperexcitability. Conclusions: Significant motor unit loss measured by MUNIX is already present at the earliest detection of muscle weakness in ALS. Increased MUSIX and altered axonal physiology are associated with axonal sprouting and geometry change(1), along with ion channel dysfunction(2). Future trials targeting muscle weakness in ALS should consider the altered neuronal physiology during early disease stages and utilize neurophysiological biomarkers only in normal-to-mildly weak muscles.