Background: Large artery stiffness is associated with fitness phenotypes in adulthood, but the association of childhood physical fitness with adult arterial stiffness is unknown. Aim: To determine the longitudinal association of childhood physical fitness phenotypes with adult carotid artery stiffness. Methods: Data were from 2,008 participants in the Childhood Determinants of Adult Health Study (52% female) with baseline measurements collected in 1985 when aged 7-15 years and follow-up measurements collected 20 years later. Fitness measures included muscular strength (isometric handgrip, shoulder, and leg strength), power (standing long jump), endurance (number of push-ups in 30 seconds), cardiorespiratory fitness (physical work capacity at heart rate of 170 bpm [PWC170] and time to complete a 1.6km run), and flexibility (sit and reach distance). All predictors were expressed as age- and sex-specific z-scores. Arterial stiffness was measured as carotid distensibility (CD) by ultrasound. Results: In univariable analysis, no association was found between childhood muscular strength (β=-0.029), power (β=-0.013), endurance (β=0.00051), PWC170 (β=0.00045), 1.6km run: (β=-0.0077), and adult CD (all p>0.30). However, an association was present for flexibility in childhood and adult CD (β=-0.038, p=0.028). Following adjustment for relevant adult measures, age and sex, no childhood physical fitness measures were associated with adult CD (muscular strength: β=-0.044; power: β=0.0077, endurance: β=0.017; PWC170: β=-0.0010; 1.6km run: β=0.0058, flexibility: β=-0.035 all p>0.20). Conclusion: Measures of physical fitness in childhood were weakly associated with adult arterial stiffness. Given the reported relationship of adult fitness phenotypes with arterial stiffness, our data suggest current fitness or activities associated with current fitness might play a greater role in determining adult arterial stiffness.