Skeletal muscle plasticity involves the transition of muscle fibers through different structural and metabolic phenotypes, to an endpoint matching environmental conditions. The result could be an increase of muscles in mass and performance, as in the case of exercise, or a functional decline, as in the case of age-dependent sarcopenia. We have recently obtained the proteome of single mouse muscle fibers using a liquid chromatography/mass spectrometry-based workflow optimized for low abundant and high dynamic range samples (Murgia et al, EMBO Reports 2015). We here analyse by shotgun proteomics single human muscle fibers from biopsies of healthy subjects differing in age and daily physical activity, as well as of patients with limited mobility. Our results indicate that the proteomes of different fiber types can be clearly distinguished based on structural and metabolic features. Additionally, activity-dependent proteomic features segregate patients’ muscle fibers from those of physically active subjects. Our results will provide important insight into human skeletal muscle plasticity at the level of its cellular units.