BACKGROUND Physical activity performed at moderate intensity is associated with reduced risk of mortality, cardiovascular disease, hypertension, and some types of cancers. However, vigorous physical activity during participation in college athletics may increase the risk of injury, which might limit future physical activity levels. PURPOSE To evaluate differences in current physical fitness levels between former Division I athletes and noncollegiate athletes. STUDY DESIGN Cross-sectional study. LEVEL OF EVIDENCE Level 3. METHODS The sample was recruited from a large midwestern university alumni database and consisted of 2 cohorts: (1) former Division I athletes (n = 100; mean age, 53.1 ± 7.4 years) and (2) nonathletes who were active in college (n = 100; age, 51.4 ± 7.3 years). Individuals answered a demographics questionnaire and completed a physical fitness assessment consisting of 7 measures: percent body fat, 1-mile walk, sit-to-stand test, push-up, half sit-up test, sit and reach test, and back scratch test. RESULTS Performance was significantly worse for former Division I athletes compared with nonathletes for percent body fat (mean difference, 7.58%; F(1, 198) = 59.91; P < 0.01), mile time (mean difference, 2.42 minutes; F(1, 198) = 1.74; P = 0.03), sit-to-stand test (mean difference, 4.3 repetitions; F(1, 198) = 6.59; P = 0.01), and push-up test (mean difference, 8.9 repetitions; F(1, 198) = 7.35; P = 0.01). CONCLUSION Former Division I athletes may be limited because of previous injury, inhibiting their ability to stay active later in life. CLINICAL RELEVANCE It is imperative that clinicians, coaches, and strength and conditioning specialists understand the possible future repercussions from competing at the Division I level.