Regular physical exercise induces cardiac adaptations that can overlap pathological conditions. Controversy still persists about the variability of myocardial deformation in different types and intensity of exercise. The aim of this study was to assess myocardial longitudinal deformation in athletes with different level of exercise. Two groups of young athletes involved in endurance sports characterized by high intensity dynamic component were enrolled. According to the level and the number of exercise training hours/week, two groups were defined: Group 1-high level (national/international and ≥ 20 training-hours/week; N = 60); Group 2-low level (recreational/regional and < 10 training-hours/week; N = 48). A comprehensive transthoracic echocardiogram including evaluation of global longitudinal strain (GLS) assessed by 2D speckle-tracking was performed. Athletes in Group 1 showed more pronounced cardiac remodeling and enhanced diastolic function. No significant differences were evident in left ventricle ejection fraction (LVEF) between groups. Overall, GLS (absolute values) was 18.0 ± 2.5%, but significantly lower in Group 1 compared to Group 2 (17.3 ± 2.6% vs. 18.9 ± 2.1%; p = 0.001). Thirty-three (31%) athletes had GLS below 17%, more frequently in Group 1 (79% vs. 45%; p = 0.001), with higher LV and left atrium volumes, lower E wave and A wave peak velocities and E/e′ ratio. In a multivariate analysis to belong to Group 1 was the only independent variable associated with GLS < 17% (OR 6.5; 95% CI 2.4–17.4; p < 0.001). The athletes with a GLS < 17% were all men, more frequently involved in high level exercise, with higher chamber volumes and lower E/e′ ratio. Left ventricular global myocardial longitudinal deformation evaluated by GLS was significantly lower in athletes with higher level of exercise. Although GLS in athletes overlap several pathological conditions, these lower values are associated with an enhanced diastolic performance that allows discrimination between physiologic adaptations and pathology.