Stretch of myocardium improves contractility by enhanced cross-bridge cycling, which increases the energy demand. Adaptation of the energy metabolism would enable the myocardium to cope with the elevated energy demand. Little is known about such alterations and the trigger mechanisms involved. We assessed proteome changes at 6h after acute increase of preload in isolated rabbit myocardium. Right ventricular papillary muscles remained unloaded or were stretched to a passive tension of 3mN/mm2. All preparations performed isotonic contractions triggered by stimulation at 1 Hz. Proteome changes were analyzed using 2D-gel electrophoresis and LC MS/MS. 30 proteins were upregulated after 6h of preload (>1.5-fold regulation, p<0.05, n=6), of which 15 were identified. Several enzymes involved in both glucose and fatty acid metabolism (e.g. aconitase, pyruvate kinase, fructose bishosphate aldolase, ATP synthase alpha chain, acetyl-CoA acetyltransferase, ubichinol cytochrome C reductase, hydroxyacyl-CoA dehydrogenase) were upregulated. Upon pretreatment with the calcineurin (CaN) inhibitor cyclosporine A (CsA, 1mmol/L) none of these proteins was upregulated by preload. Preload in isolated rabbit myocardium upregulates enzymes important for ATP production, which is abolished by inhibition of CaN activity. Thus, adaptation of myocardial energy metabolism to increased load critically depends on CaN activation.