INTRODUCTION: Astronauts have sustained musculoskeletal injuries and minor trauma in space, but our knowledge of these injuries is based mainly on anecdotal reports. The purpose of our study was to catalog and analyze all in-flight musculoskeletal injuries occurring throughout the U.S. space program to date. METHODS: A database on in-flight musculoskeletal injuries among U.S. astronauts was generated from records at the Johnson Space Center. RESULTS: A total of 219 in-flight musculoskeletal injuries were identified, 198 occurring in men and 21 in women. Incidence over the course of the space program was 0.021 per flight day for men and 0.015 for women. Hand injuries represented the most common location of injuries, with abrasions and small lacerations representing common manifestations of these injuries. Crew activity in the spacecraft cabin such as translating between modules, aerobic and resistive exercise, and injuries caused by the extravehicular activity (EVA) suit components were the leading causes of musculoskeletal injuries. Exercise-related injuries accounted for an incidence of 0.003 per day and exercise is the most frequent source of injuries in astronauts living aboard the International Space Station (ISS). Interaction with EVA suit components accounted for an incidence of 0.26 injuries per EVA. DISCUSSION: Hand injuries were among the most common events occurring in U.S. astronauts during spaceflight. Identifying the incidence and mechanism of in-flight injuries will allow flight surgeons to quantify the amount of medical supplies needed in the design of next-generation spacecraft. Engineers can use in-flight injury data to further refine the EVA suit and vehicle components.