Left atrial strain (LAS) assessment by speckle tracking echocardiography (STE) has been shown to be a remarkable means of quantifying LA function as an early marker of LV pathology. As exercise testing is also performed on a treadmill, the aim of this study was to investigate the effect of upright posture on LAS in healthy athletes. Fifty male athletes (mean age 25.7 ± 7.3 years) underwent transthoracic echocardiography (TTE) in the upright and left lateral positions. In addition to the conventional echocardiographic parameters, in all athletes, LA conduction strain (LAScd), contraction strain (LASct), reservoir strain (LASr), and maximum LA volume (LAVmax) were assessed by STE in both positions. Comparing upright posture and the left lateral position, LAScd (-14.0 ± 5.9% vs. -27.4 ± 7.1%; p < 0.001), LASct (-4.6 ± 3.5% vs. -11.3 ± 4.1%; p < 0.001), LASr (18.7 ± 7.6% vs. 38.7 ± 8.0%; p < 0.001), and LAVmax (24.4 ± 8.8% vs. 50.0 ± 14.2%) differed significantly. Upright posture has a significant effect on LA deformation, with decreased LAScd, LASct, and LASr. The results of this study contribute to the understanding of athletes' hearts and must be considered when performing echocardiography in healthy athletes on a treadmill.