INTRODUCTION: Exposure to microgravity reduces muscle mass, volume, and performance. The ingestion of protein, especially combined with carbohydrate intake and exercise after ingestion, improves net muscle protein synthesis and increases muscle mass. However, there are few studies on this relationship during and after a long-term spaceflight. The objective of this study was to investigate the influence of protein and the combined effects of carbohydrate intake on muscle performance following long-term spaceflight.METHODS: This study is a retrospective cohort study involving secondary analysis of data stored in the NASA Lifetime Surveillance of Astronaut Health Repository. Multivariable analysis was performed to evaluate the impact of protein intake on physical performance by considering covariates potentially associated with each model.RESULTS: After adjusting for sex, age, flight week, energy intake, and preflight physical performance, protein intake was found to be significantly associated with concentric measurements for knee extension ( 51.66), ankle plantar flexion ( 32.86), and eccentric measurements for ankle plantar flexion ( 79.85) at 5 d after landing. Significant associations remained after controlling for exercise effect. No significant interaction between protein and carbohydrate intake was observed in either model.DISCUSSION: Protein intake during spaceflight was related to physical performance for knee extension and ankle plantar flexion, even after taking exercise effect into consideration. However, protein and carbohydrate intake provided no synergetic benefit. This suggests that high protein intake, about twice the current average intake, may serve as a countermeasure to offset the negative effects of long-duration spaceflights.Nozawa Y, Wagatsuma Y. Protein intake and physical performance following long-term stay on the International Space Station. Aerosp Med Hum Perform. 2021; 92(3):153159.