Abstract Context Anabolic hormones are important factors in maintaining muscle mass for aging men, but their role in overall motor unit structure and function is unclear. Objective The objective of this work is to determine associations of anabolic and reproductive hormone levels with motor unit characteristics in quadriceps muscle in older healthy and frail men. Design This work is an observational cohort study of community-dwelling men. Participants Participants included healthy and frail men younger than 65 years. Intervention No intervention was performed. Outcome measure Quantitative assessments of electromyography-derived motor unit potential size (MUP) and compound muscle action potential size (CMAP) of the vastus lateralis muscle. Results We studied 98 men (mean ± SD: age 73 ± 6 years; body mass index [BMI] 25.7 ± 4.0 kg/m2; diabetes 11%) of whom 45% were prefrail and 18% frail. After adjusting for age, BMI, and prevalent diabetes, higher total and free testosterone levels were significantly related to larger CMAP (total testosterone: β [95% CI]: 0.3 [0.08-0.53]; free testosterone: 0.34 [0.13-0.56]). Exploratory analysis showed the relationship between free testosterone and CMAP was stronger in frail rather than robust men. In univariate analyses, estradiol was associated with CMAP size (0.37 [0.16-0.57]); and vitamin D was associated with MUP size (0.22 [0.01-0.43]) but these relationships were no longer significant after adjusting for potential confounders. Conclusion Our data highlight the associations between androgen levels and the electrophysiological characteristics of older men, particularly in the frail. Clinical trials involving administration of androgens will help to elucidate the potential benefits of intervention on neuromuscular function and/or frailty status.