In order to study myocardial hypertrophy under in vitro condtions, we developed an experimental system and protocol in which mechanical conditions of isolated multicellular myocardium can be controlled while function can be continuously assessed. This in vitro culture system now allows us to investigate how mechanical overload impacts on cardiac hypertrophy in absence of systemic factors. In this system, small right ventricular rabbit trabeculae were subjected to different modes of mechanical load, while being electrically stimulated to contract at 1 Hz at 37 °C. Muscles subjected to prolonged isometric contractions at high, but physiological, pre- and after-load showed a rapid induction of cardiac hypertrophy; overall muscle diameter increased by 4.3±1.4 and 17.9±4.0% after 24 and 48 hours, respectively. This finding was confirmed at the cellular level; individual myocyte width significantly increased after 24 and 48 hours. In muscles subjected to a low pre-load, or in absence of after-load, this hypertrophic response was absent. Functionally, after 24 hours of isometric contractions at high load, active developed tension had gradually increased to 168±22% of starting values. Proteomic analysis of this cultured myocardium demonstrated reproducible changes in the protein expression pattern, and included an up-regulation of myofilament proteins, myosin light chain isoforms, alpha-b crystalline, and breast cancer 1 protein, and a down-regulation of myoglobin. We conclude that multicellular myocardium can be stressed to undergo rapid hypertrophy in vitro, and changes in function and protein expression can be investigated during the transition from healthy myocardium to early hypertrophy.