ImportanceAlthough research indicates that low fitness in youth is associated with a higher risk of chronic disability in men, the association of fitness in adolescence with work ability in working men and women remains unknown.ObjectiveTo examine the associations of adolescent health-related physical fitness with future work ability.Design, Setting, and ParticipantsThis 45-year observational cohort study, conducted in Finland, examined the direct and indirect longitudinal associations of objectively measured health-related fitness in adolescence (in 1976) with self-reported work ability and sickness absence in early middle age (in 2001) and the Work Ability Index in late middle age (in 2021). A countrywide stratified random baseline sample included fitness measurements for cardiorespiratory fitness (CRF) (running 1.5 km for girls and 2 km for boys), musculoskeletal fitness (MF; standing broad jump and sit-ups for both sexes, pull-ups for boys, and flexed-arm hang for girls), and height and weight, from which body mass index (BMI) was calculated. Structural equation modeling–based path analysis, adjusted for age and sex at baseline and for educational level, work-related physical strain, and leisure-time physical activity in late middle age, was conducted. Data analysis was performed from January to July 2023.Main Outcomes and MeasuresSelf-reported work ability was measured with structured questions in early middle age and with the validated Work Ability Index in late middle age.ResultsThe final sample from longitudinal analyses (1207 individuals; 579 [48%] male individuals) consisted of participants with fitness measurement from age 12 to 19 years, and work ability assessment from age 37 to 44 years and/or age 57 to 64 years. Higher adolescent CRF was associated with higher work ability (839 participants; β = 0.12; 95% CI, 0.01 to 0.22; P = .03) and lower sickness absence (834 participants; β = −0.07; 95% CI, −0.12 to −0.02; P = .004) in early middle age and, indirectly, mediated by work ability in early middle age, with a higher work ability at the end of working age (603 participants; β = 0.04; 95% CI, 0.001 to 0.08; P = .04). The results remained consistent in both sexes and after adjustment for the confounders. Neither MF nor BMI was associated with work ability (MF, 1192 participants; β = −0.07; 95% CI, −0.17 to 0.03; BMI, 1207 participants, β = 0.09; 95% CI, −0.004 to 0.19) or sickness absence (MF, 1185 participants, β = 0.02; 95% CI, −0.03 to 0.06; BMI, 1202 participants, β = −0.03; 95% CI, −0.09 to 0.03) in early middle age or with late middle age work ability, mediated by work ability in early middle age (MF, 603 participants, β = −0.02; 95% CI, −0.06 to 0.01; BMI, 603 participants, β = 0.03; 95% CI, −0.004 to 0.07).Conclusions and RelevanceThese findings suggest that low CRF in youth is associated with poor work ability at the middle and end of working life, which highlights the informative and prognostic value of CRF assessment early in youth. Enhancing CRF in the first decades of life might contribute to better work capacity and productivity in the labor force, which would have implications for health, quality of life, society, and the economy.