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Aims: Recent advancements in echocardiographic technology allow to analyse myocardial strain in multiple layers. Little is known about the impact of age on layer-specific longitudinal strain in healthy subjects. The aim of this study was to analyse the influence of age on multilayer longitudinal strain and establish normal reference values of layer-specific strain according to age decades in a healthy population referring to our echo laboratory using 2D speckle-tracking echocardiography with layer-specific software., Methods and Results: Two-hundred sixty-six healthy, consecutive subjects (mean age = 39.2 ± 17.5 years, women/men = 137/129), free of cardiovascular risk factors, were enrolled. Subjects were divided according to six age decades: 10-19, 20-29, 30-39, 40-49, 50-59, >60 years. All subjects underwent a complete echo Doppler examination including quantitation of 2D global longitudinal strain (GLS). Subendocardial longitudinal strain (LSsubendo), subepicardial longitudinal strain (LSsubepi), and strain gradient (LSsubendo - LSsubepi) were also determined. GLS (P < 0.001), LSsubendo, and LSsubepi (both P < 0.0001) were all progressively reduced with increasing age decades, but post hoc intra-group analyses demonstrated that the decline of GLS, LSsubendo, and LSsubepi was significant in the decades 50-60 and ≥60 years. In separate multiple linear regression analyses, the effect of age on GLS, LSsubendo, and LSsubepi remained significant even after adjusting for clinical and echocardiographic confounders. Strain gradient remained unchanged in age decades., Conclusion: Ageing shows an independent effect on GLS, LSsubendo, and, particularly on, LSsubepi. Our data also provide normal reference values of layer-specific longitudinal strain for age decades.