Background Protein supplementation can enhance recovery of exercise-induced muscle damage in young adults. It is currently unknown whether this could be extrapolated to older adults who have a disturbed muscle protein synthetic response. The aim of this study was to assess whether protein supplementation could attenuate exercise-induced muscle damage and soreness after prolonged moderate-intensity walking exercise in older adults.Methods In a double-blind, placebo-controlled intervention study, 104 participants (81% male, BMI: 26.5±2.5 kg/m 2 ) of 65 years or older used either a protein (n=50) or placebo supplement (n=54) during breakfast and directly after exercise cessation. Study participants walked 30/40/50 km per day on 3 consecutive days. Muscle soreness and fatigue were determined at baseline, and after the 1 st and 3 rd exercise day with a numeric rating scale. Blood samples, to measure plasma creatine kinase (CK) concentrations and serum inflammation markers, were obtained at baseline (pre-exercise), and after the 1 st and 3 rd exercise day.Results Habitual protein intake was comparable between the protein (0.92±0.27 g/kg/d) and placebo group (0.97±0.23 g/kg/d, P= 0.31). At baseline, comparable CK concentrations were found between the protein and the placebo group (110 (IQR: 84–160 U/L) and 115 (IQR: 91–186 U/L), respectively, P= 0.84). Prolonged walking (protein: 32±9 km/d, placebo: 33±6 km/d) resulted in a cumulative increase of CK in both the protein (∆283 (IQR: 182–662 U/L)) and placebo group (∆456 (IQR: 209–885 U/L)) after three days. CK elevations were not significantly different between groups ( P= 0.43). Similarly, no differences in inflammation markers, muscle soreness and fatigue were found between groups.Conclusions In contrast to findings in young adults, protein supplementation does not attenuate exercise-induced muscle damage, muscle soreness or fatigue in older adults performing prolonged moderate-intensity walking exercise.