Background Standard apical four-chamber and two-chamber views often maximize the long-axis of the left ventricle, resulting in artifactitious foreshortening of the left atrium (LA), which may overestimate LA longitudinal reservoir strain (LALS). We compared LALS values between 2D echocardiography (2DE) and 3D echocardiography (3DE) in healthy subjects to determine whether 2DE speckle tracking analysis overestimates the reference value of LALS. Methods and results In this study, 4 types of cohorts were included: 1. 105 normal subjects (retrospectively), 2. 53 patients with cardiovascular diseases (retrospectively), 3. 15 patients who received cardiac magnetic resonance (prospectively), and 4. 20 normal subjects (prospectively). LALS and LA length were measured using both 2DE and 3DE in 105 healthy subjects (median age: 42 years). Biplane LALS was measured in apical four- and two-chamber views using 2DE speckle tracking software, and 3DE LALS was measured using new 3DE LA strain software. To determine sensitivity, we also performed the same analysis in 53 patients with cardiovascular disease. The mean value of biplane LALS was 39.6%. LA length at both end-diastole (r = -0.43) and end-systole (r = -0.54) was negatively correlated with biplane LALS. Multivariate regression analysis revealed that both end-diastolic and end-systolic LA length had significant negative relationships with biplane LALS after adjusting for anthropometric and echocardiographic image quality parameters. 3DE LALS (23.7±7.6%) gave significantly lower values than 2DE LALS (39.5±12.0%, p<0.001) with a weak correlation (r = 0.33). LA length measured by 2DE was significantly shorter than that measured by 3DE. The same trend was observed in diseased patients. Conclusions Our results revealed that in 2DE, the LA cavity consistently appears longitudinally foreshortened in apical views, potentially overestimating LALS. 3DE may overcome this limitation.