The shiitake (Lentinus edodes) extract is purported to have potent antioxidant, anti-inflammatory and regenerative properties due to presence of many bioactive compounds such as ergothioneine. This study was designed to assess the antioxidant and anti-inflammatory activity of shiitake extract in healthy men exposed to exercise-induced skeletal muscles damage. Subjects ingested shiitake mushroom extract (700 mg, two times per day) or placebo for 10 days prior to two separate exercise trials (crossover study). The exercise session involved 90 min run at 65% VO2max (0% gradient) and 15-min eccentric phase at 65% VO2max (-10% gradient). Subjects experienced creatine kinase (peak 461±206 IU/L) and leukocytes (peak 9.82 x 103/μL) elevations indicating muscle damage and inflammation. Exercise altered plasma IL-6 (peak 5.29±0.78 pg/mL), IL-10 (peak 24.75±6.22 pg/mL) and IL-1β (peak 0.54±0.09 pg/mL) levels but did not affect tumour necrosis factor α (TNF-α) level relative to baseline. Shiitake extract did not demonstrate any effect on immune cells number and inflammatory mediators level, with the exception of IL-10. Thiol redox status (GSHtotal-2GSSG/GSSG) and niric oxide (NO) concentration increased after shiitake extract whereas H2O2 and 8-isoprostanes did not change. In conclusion, shiitake mushroom extract had no effect on markers of inflammation following prolonged eccentric exercise but demonstrated an antioxidant activity through the regulation of nitric oxide concentration and thiol redox status.