The decline in skeletal muscle regenerative capacity with age is partly attributed to muscle stem cell (satellite cell) dysfunction. Recent evidence has pointed to a strong interaction between myoblasts and fibroblasts, but the influence of age on this interaction is unknown. Additionally, while the native tissue environment is known to determine the properties of myogenic cells in vitro, how the aging process alters this cell memory has not been established at the molecular level. We recruited 12 young and 12 elderly women, who performed a single bout of heavy resistance exercise with the knee extensor muscles of one leg. Five days later, muscle biopsies were collected from both legs, and myogenic cells and nonmyogenic cells were isolated for in vitro experiments with mixed or separated cells and analyzed by immunostaining and RT-PCR. A lower myogenic fusion index was detected in the cells from the old versus young women, in association with differences in gene expression levels of key myogenic regulatory factors and senescence, which were further altered by performing exercise before tissue sampling. Coculture with nonmyogenic cells from the elderly led to a higher myogenic differentiation index compared with nonmyogenic cells from the young. These findings show that the in vitro phenotype and molecular profile of human skeletal muscle myoblasts and fibroblasts is determined by the age and exercise state of the original in vivo environment and help explain how exercise can enhance muscle stem cell function in old age.