Background: Myocardial stress and strain are considered primary mechanical stimuli for hypertrophic remodeling. Their values and significance in the intact beating heart during chronic overload remain poorly characterized. Methods and results: Left-ventr icular (LV) dimensions (echocardiography) and pressure (invasive) were simultaneously recorded in anesthetized dogs at sinus rhythm (SR), acute and 1, 2, 6, 12 weeks of atrioventricular block (AVB), leading to structural, electrical and contractile remodeling. Mechanical load of the myocardium was quantified as myofiber stress (σf), being force along myofiber orientation per cross-sectional area, and natural myofiber strain (ef), being change in natural logarithm of myofiberlength (l) divided by its reference length: ef = ln(l/lref). Time courses of σf and ef were calculated from LV pressure and dimensions, using a validated mathematical model of cardiac mechanics. End-diastolic σf increased from 2.0 ±0.1 kPa at SR to 3.4 ± 0.3 kPa at acute AVB, remaining elevated for > 6 weeks. Systolic σf was not affected by AVB. Ejection strain rose instantly upon AVB, reaching a maximum at 2 weeks: 0.24 ± 0.02 vs. 0.10 ± 0.01 at SR. The increase of myofiber stroke work (σf-ef loop area) from 3.1 ± 0.3 at SR to 6.0 ± 0.5kJ/m3/beat at 1 week AVB was attributed mainly to an increase of strain during ejection. Stroke work and ejection strain remained elevated up to 12 weeks. The rate of LV-mass increase was maximal (2.2 ± 0.4 g/day) at 1 week AVB. Conclusions: Serial mechanical phenotyping is feasible in the intact anesthetized dog with chronic ventricular overload. Our new approach yields values of mechanical load that are comparable to those found in isolated myocardium by others. In chronic AVB, both end-diastolic myofiber stress and ejection strain are increased. Early increases of both parameters coincide with peak hypertrophic growth, suggesting their important role for mechanotransduction. Peak systolic σf is likely not important for hypertrophy in this model, since it does not change throughout the experiment.