INTRODUCTION: Regional heterogeneity of the human heart plays an important role in left (LV) and right (RV) ventricular function, and may contribute to enhanced myocardial efficiency in the athlete's heart. PURPOSE: This study comprehensively characterized regional and transmural myocardial tissue deformation (strain) in recreationally active and endurance trained men to determine if regional non-uniformity evolves alongside morphological adaptations associated with endurance training. METHODS: Echocardiography was used to measure LV and RV global, regional (apical, mid, basal) and transmural (endocardial, epicardial) longitudinal strain in 30 endurance-trained (ET; age: 31±2yr; BMI: 23.1±0.5kg/m; VO2peak: 60.2±6.5mL/kg/min) and 30 recreational-active men (RA; age: 29±2yr; BMI: 23.4±0.4kg/m; VO2peak: 42.6±4.6mL/kg/min). Non-uniformity was characterized using apex-to-base and transmural (endocardial-to-epicardial) strain gradients. RESULTS: Global longitudinal strain was similar in ET and RA in the left (-17.4±0.4 vs -17.8±0.5%, p=0.662) and right ventricle (-25.8±0.8 vs 26.4±1.0%, p=0.717). The apex-to-base strain gradient was greater in ET than RA in the left (-6.5±0.7 vs -2.7±0.8%, p=0.001) and right ventricle (-9.6±1.8 vs -3.0±1.6%, p=0.010). The LV transmural strain gradient was greater than RV in both groups, but similar in ET and RA (-4.7±0.2 vs -4.7±0.2%, p=0.850), while RV transmural strain gradient was greater in ET than RA (-3.4±0.3 vs -1.6±0.4%, p=0.003). RV apex-to-base and transmural strain gradients correlated with RV end-diastolic area (R=0.536 & 0.555, respectively, p<0.01) and VO2peak (R=0.415 & 0.677, respectively, p<0.01). CONCLUSION: Transmural non-uniformity is more pronounced in the left ventricle than the RV free wall; however, RV functional non-uniformity develops markedly following endurance training. Differences in myocardial architecture and exercise-induced wall stress in the left and right ventricles are possible explanations for the marked functional non-uniformity throughout the myocardium and in response to endurance exercise training.