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Protease supplementation has been purported to reduce the damaging effects of eccentric exercise and accelerate recovery of muscle function, possibly by regulating inflammation.To determine the effectiveness of protease supplementation in attenuating eccentric exercise-induced skeletal muscle damage and inflammation.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 V˙O2max 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 V˙O2max. 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.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.Protease supplementation seems to attenuate muscle strength losses after eccentric exercise by regulating leukocyte activity and inflammation.