The effect of two running sessions completed within a 12-h period on hemolysis, inflammation, and hepcidin activity in endurance athletes was investigated. Ten males completed two experimental trials in a randomized, counterbalanced order. The two trials included (a) a one-running-session trial (T1) including 10 x 1 km interval repeats (90% peak VO2 velocity), and (b) a two-running-session trial (T2), comprising a continuous 10-km run (70% peak VO2 velocity), and a 10 x 1 km interval run (90% peak VO2 velocity) completed 12 h later. Interleukin-6 (IL-6), free hemoglobin (Hb), haptoglobin (Hp), iron, ferritin, and hepcidin were assessed post-exercise. After the T1 and T2 interval runs, free Hb was significantly increased and Hp significantly decreased (p <or= 0.05), with a cumulative effect shown in T2 after the second run (p <or= 0.05). The IL-6, serum iron, ferritin, and hepcidin activity were increased after each running session (p <or= 0.05), with no cumulative effect in T2. In conclusion, a cumulative effect of two running sessions on hemolysis was shown, but no similar effect with inflammation and hepcidin activity was evident.