Twelve weanling Quarter Horses were divided randomly into 2 groups; exercised (EX) and non-exercised (NONEX). The EX horses were divided randomly into 2 groups subjected to either a 15-day or 30-day exercise protocol and weanlings were exercised at a brisk trot (3 m/s) on a high speed treadmill. Muscle satellite cells were obtained by surgical biopsy from the biceps femoris at the conclusion of exercise protocols. Time course studies were used to examine the main effects of exercise on proliferation and differentiation of equine satellite cells (ESC) in vitro. No differences occurred between proliferation and differentiation of cells from 15- or 30-day EX and NONEX treatments. In a second experiment, the ability of serum from EX or NONEX horses to stimulate proliferation or differentiation of ESC in vitro was examined. Serum had a significant effect on both proliferation and differentiation. During Days 3 and 5 in culture, serum from EX weanling horses increased (P<0.005) total cell numbers; however, after 7 days, no differences (P>0.10) existed between serum sources. From Days 3–7, satellite cells cultured in serum from EX horses had greater (P<0.005) per cent fusion than cells cultured in serum from NONEX weanling horses. Origin of satellite cell cultures (EX or NONEX) caused no differences. A constituent seems to be present in the serum from EX that is capable of stimulating increased levels of proliferation and differentiation of ESC.