OBJECTIVE: The threshold tracking technique is a new approach to non-invasively assess biophysical properties of axonal membrane in human subjects. The aim of this study was to evaluate the effects of age and gender on excitability properties of human motor axons. METHODS: Computerized threshold tracking was used to measure multiple excitability indices in median motor axons of 93 normal subjects (50 men; age, 20-86 years). RESULTS: Regression analyses showed that the higher age was associated with longer strength-duration time constant (p=0.03), smaller threshold changes in depolarizing threshold electrotonus (p=0.02), smaller supernormality (p=0.01), and steeper slope of the current-threshold relationship for hyperpolarizing currents (p<0.001). There were slight sex differences in rheobase, threshold electrotonus, supernormality, late subnormality, and current-threshold slope, though they were significant only in the subgroup with age <50 years. CONCLUSIONS: Aging may increase persistent sodium currents, inward rectification, and possibly, outward potassium currents. The combination of changes raises the possibility of slight membrane depolarization in elderly people. For the sex-related differences, further studies will be required with the evaluation of sex hormonal effects. SIGNIFICANCE: Age-related effects on excitability properties are subtle, but should be taken into consideration in the clinical application of nerve excitability testing, particularly in elderly subjects.