Objectives. We examined whether diastolic left ventricular function in young and senior lifelong endurance runners was significantly different from that in sedentary age-matched controls, and whether lifelong endurance running appears to modify the age-related decline in diastolic left ventricular function. Design. The study comprised 17 senior athletes (age: 59–75 years, running distance: 30–70 km/week), 10 young athletes (age: 20–36 years, matched for running distance), and 11 senior and 12 young weight-matched sedentary controls. Peak early (E) and late (A) mitral inflow and early (e’) and late (a’) diastolic and systolic (s’) annular longitudinal tissue Doppler velocities were measured by echocardiography during four stages (rest, supine bike exercise at 30% and 60% of maximal workload, and recovery). Results. The athletes had marked cardiac remodeling, while overall differences in mitral inflow and annular tissue Doppler velocities during rest and exercise were more associated with age than with training status. The senior participants had lower E/A at rest, overall lower E, e’ and s’, and greater E/e’ compared to the young participants (all values of P < 0.05). The athletes had greater E/A (P = 0.004), but tissue Doppler velocities were not different from those of the controls. Conclusions. Lifelong endurance running was not found to be associated with major attenuation of the age-related decline in diastolic function at rest or during exercise.