Protease supplementation has been purported to reduce the damaging effects of eccentric exercise and accelerate recovery of muscle function, possibly by regulating inflammation. Purpose: To determine the effectiveness of protease supplementation in attenuating eccentric exercise-induced skeletal muscle damage and inflammation. Methods: After standard physical and hemodynamic assessment and fasting venous blood samples, subjects performed isokinetic extension/flexion of the quadriceps group on a Biodex isokinetic dynamometer at 60°•s-1, followed by V̇O2max testing. Subjects were randomly assigned to consume 5.83 g daily of either a cellulose placebo (N = 15; 22.27 ± 3.33 yr, 71.17 ± 2.91 inches, 179.4 ± 24.05 lb, 50.55 ± 5.66 mL•kg-1•min-1) or a proteolytic supplement containing fungal proteases, bromelain, and papain (N = 14; 22.85 ± 5.9 yr, 70.0 ± 2.67 inches, 173.11 ± 29.94 lb, 49.69 ± 6.15 mL•kg-1•min-1) for a period of 21 d. After the supplementation period, subjects donated blood samples before performing a 45-min downhill (-17.5%) treadmill protocol at 60% of V̇O 2max. An additional four blood draws and three muscle function tests were performed during the next 48 h. Blood was analyzed using standard hematology and clinical chemistry, enzyme-linked immunosorbent assay, and bead array. Blood data were analyzed using multivariate analysis of variance (MANOVA) with repeated measures, whereas Biodex data were analyzed using a MANOVA on %Δ values. Results: Significant group differences (T1-T3, P = 0.033; T1-T4, P = 0.043) and another strong trend (T1-3 h, P = 0.055) were observed for flexion (peak torque %Δ at 60°•s-1) indicating higher force production in the protease group. Significant group × time interactions (P < 0.05) were observed, including elevations in circulating eosinophils and basophils in the protease group coinciding with lower levels of serum cyclooxygenase 2, interleukin 6, and interleukin 12 in this group. Conclusions: Protease supplementation seems to attenuate muscle strength losses after eccentric exercise by regulating leukocyte activity and inflammation. © 2009 by the American College of Sports Medicine.