Purpose: This study aimed to examine the changes in skeletal muscle (SM) alpha-actin, myoglobin (Mb) and hydroxyproline (HP) in plasma and other indirect markers of muscle damage after repeated bouts of eccentric cycling. Methods: Ten healthy men (23.3 +/- 2.8 years) performed two 30-min eccentric cycling bouts at 100% of maximal concentric power output (230.7 +/- 36.9 W) separated by 2 weeks (ECC1 and ECC2). Maximal voluntary isometric contraction (MVIC) peak force of the knee extensor muscles, muscle soreness (SOR), pain pressure threshold (PPT) and plasma levels of SM alpha-actin, Mb, and HP were measured before, 0.5, 3, 24-168 h after each cycling bout. Results: MVIC peak force decreased on average 10.7 +/- 13.1% more after ECC1 than ECC2. SOR was 80% greater and PPT was 12-14% lower after ECC1 than ECC2. Plasma SM alpha-actin levels increased at 0.5, 3, and 24-72 h after ECC1 (26.1-47.9%), and SM alpha-actin levels at 24 h after ECC1 were associated with muscle strength loss (r = -0.56, P = .04) and SOR (r = 0.88, P = .001). Mb levels increased at 0.5, 3, and 24 h after ECC1 (200-502%). However, Mb levels at 24 h after ECC1were not associated with muscle strength loss and SOR. HP levels remained unchanged after ECC1. ECC2 did not increase SM alpha-actin, Mb and HP levels. Conclusion: Our results indicate that alpha-actin could be used as a potential marker for the early identification of SM damage due to its early appearance in plasma and its association with other indirect markers of muscle damage.