Purpose: In girls and women, the authors studied the effects of an acute bout of low-impact, moderate-intensity exercise serum on myoblast and osteoblast proliferation in vitro. Methods: A total of 12 pre/early pubertal girls (8–10 y old) and 12 women (20–30 y old) cycled at 60% VO2max for 1 hour followed by 1-hour recovery. Blood samples were collected at rest, mid-exercise, end of exercise, mid-recovery, and end of recovery. C2C12 myoblasts and MC3T3E1 osteoblasts were incubated with serum from each time point for 1 hour, then monitored for 24 hours (myoblasts) or 36 hours (osteoblasts) to examine proliferation. Cells were also monitored for 6 days (myoblasts) to examine myotube formation and 21 days (osteoblasts) to examine mineralization. Results: Exercise did not affect myoblast or osteoblast proliferation. Girls exhibited lower cell proliferation relative to women at end of exercise (osteoblasts, P = .041; myoblasts, P = .029) and mid-recovery (osteoblasts, P = .010). Mineralization was lower at end of recovery relative to rest (P = .014) in both girls and women. Myotube formation was not affected by exercise or group. Conclusion: The systemic environment following one acute bout of low-impact moderate-intensity exercise in girls and women does not elicit osteoblast or myoblast activity in vitro. Differences in myoblast and osteoblast proliferation between girls and women may be influenced by maturation.