Background A retrospective examination was conducted of injuries, physical fitness, and their association among Federal Bureau of Investigation (FBI) new agent trainees. Methods Injuries and activities associated with injuries were obtained from a review of medical records in the medical clinic that served the new agents. A physical fitness test (PFT) was administered at Weeks 1, 7 and 14 of the 17-week new agent training course. The PFT consisted of push-ups, sit-ups, pull-ups, a 300-meter sprint, and a 1.5-mile run. Injury data were available from 2000 to 2008 and fitness data were available from 2004 to early 2009. Results During the survey period, 37% of men and 44% of women experienced one or more injuries during the new agent training course (risk ratio (women/men) = 1.18, 95% confidence interval = 1.07-1.31). The most common injury diagnoses were musculoskeletal pain (not otherwise specified) (27%), strains (11%), sprains (10%), contusions (9%), and abrasions/lacerations (9%). Activities associated with injury included defensive tactics training (48%), physical fitness training (26%), physical fitness testing (6%), and firearms training (6%). Over a 6-year period, there was little difference in performance of push-ups, sit-ups, pull-ups, or the 300-meter sprint; 1.5-mile run performance was higher in recent years. Among both men and women, higher injury incidence was associated with lower performance on any of the physical fitness measures. Conclusion This investigation documented injury diagnoses, activities associated with injury, and changes in physical fitness, and demonstrated that higher levels of physical fitness were associated with lower injury risk.