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Importance: Studies of the longevity of professional American football players have demonstrated lower mortality relative to the general population but they may have been susceptible to selection bias.Objective: To examine the association between career participation in professional American football and mortality risk in retirement.Design, Setting, and Participants: Retrospective cohort study involving 3812 retired US National Football League (NFL) players who debuted in the NFL between 1982 and 1992, including regular NFL players (n = 2933) and NFL "replacement players" (n = 879) who were temporarily hired to play during a 3-game league-wide player strike in 1987. Follow-up ended on December 31, 2016.Exposures: NFL participation as a career player or as a replacement player.Main Outcomes and Measures: The primary outcome was all-cause mortality by December 31, 2016. Cox proportional hazards models were estimated to compare the observed number of years from age 22 years until death (or censoring), adjusted for birth year, body mass index, height, and position played. Information on player death and cause of death was ascertained from a search of the National Death Index and web-based sources.Results: Of the 3812 men included in this study (mean [SD] age at first NFL activity, 23.4 [1.5] years), there were 2933 career NFL players (median NFL tenure, 5 seasons [interquartile range {IQR}, 2-8]; median follow-up, 30 years [IQR, 27-33]) and 879 replacement players (median NFL tenure, 1 season [IQR, 1-1]; median follow-up, 31 years [IQR, 30-33]). At the end of follow-up, 144 NFL players (4.9%) and 37 replacement players (4.2%) were deceased (adjusted absolute risk difference, 1.0% [95% CI, -0.7% to 2.7%]; P = .25). The adjusted mortality hazard ratio for NFL players relative to replacements was 1.38 (95% CI, 0.95 to 1.99; P = .09). Among career NFL players, the most common causes of death were cardiometabolic disease (n = 51; 35.4%), transportation injuries (n = 20; 13.9%), unintentional injuries (n = 15; 10.4%), and neoplasms (n = 15; 10.4%). Among NFL replacement players, the leading causes of death were cardiometabolic diseases (n = 19; 51.4%), self-harm and interpersonal violence (n = 5; 13.5%), and neoplasms (n = 4; 10.8%).Conclusions and Relevance: Among NFL football players who began their careers between 1982 and 1992, career participation in the NFL, compared with limited NFL exposure obtained primarily as an NFL replacement player during a league-wide strike, was not associated with a statistically significant difference in long-term all-cause mortality. Given the small number of events, analysis of longer periods of follow-up may be informative. [ABSTRACT FROM AUTHOR]