Background: College athletes participate in physical activity that may increase chronic stress and injury and induce overtraining. However, there is little known about how previous injuries that have occurred during college may limit current physical activity and/or decrease their subsequent health-related quality of life (HRQoL). Purpose: To evaluate HRQoL in former United States National Collegiate Athletic Association Division I athletes and nonathletes with the Patient-Reported Outcomes Measurement Information System (PROMIS) and a demographics questionnaire. Study Design: Cohort study (prognosis); Level of evidence, 2. Methods: The study sample was recruited through alumni databases at a large Midwestern university and consisted of 2 cohorts: (1) former Division I athletes and (2) nonathletes who participated in recreational activity, club sports, or intramurals while attending college. Participants answered a survey constructed with a web-based system. All individuals contacted were between the ages of 40 and 65 years. Study participants responded to the questions on the PROMIS scales for sleep, anxiety, depression, fatigue, pain interference, physical function, and satisfaction with participation in social roles. The PROMIS was developed to fill the void of HRQoL being evaluated by multiple instruments. An additional questionnaire was constructed to record demographic and exercise information. Results: Initially, 1280 former Division I athletes and nonathletes were contacted; 638 surveys were returned (49.8%). Surveys eligible for analysis (71.6%) were completed by 232 former Division I athletes (mean age ± SD, 53.36 ± 7.11 years) and 225 nonathletes (mean age ± SD, 53.60 ± 6.79 years). Univariate analysis for the effect of group was significantly related to PROMIS scales for physical function, depression, fatigue, sleep disturbances, and pain interference (P < .05). The overall scores were significantly worse for the former Division I athletes than for the nonathletes on 5 of the 7 scales. In addition, the former Division I athletes reported significantly more limitations in daily activities and more major and chronic injuries than did the non athlete controls. Conclusion: According to these data, former Division I athletes have decreased HRQoL compared with nonathletes. Clinical Relevance: Sports encourage physical activity, which help promote a healthy lifestyle. Moderate activity and exercise should be encouraged. However, the demands of Division I athletics may result in injuries that linger into adulthood and possibly make participants incapable of staying active as they age, thereby lowering their HRQoL.