Endurance training improves cardiac function and protects against heart disease. The rodent intensity-controlled running model replicates endurance exercise in humans and can be used to investigate molecular adaptations in the heart. Rats (n = 6, 280 +/- 3 g) performed exercise tests to measure their peak oxygen uptake (VO2peak) and training was prescribed at 70-75% VO2 peak for 30 min, 4 days/wk. Hearts were isolated 4 h after a final VO2peak test and left ventricle proteomes compared to weight-matched control animals (n = 6, 330 +/- 2 g) using differential analysis of 2-D gels. Proteins were identified by searching MS and MS/MS spectra against Swiss-Prot using MASCOT (www.matrixscience.com). Average VO2peak increased 23% (p = 0.008) over the 6-week regimen and 23 gel spots differed (p<0.05) between exercised and control hearts. Expression of myofibrillar proteins (e.g. alpha-myosin heavy chain and cardiac alpha-actin) and proteins associated with fatty acid metabolism (e.g. heart fatty acid binding protein, acetyl coenzyme A dehydrogenase and mitochondrial thioesterase-1) increased. In addition, this work discovered a novel increase in phosphorylation of heat shock protein 20 at serine 16. Previously this modification has been associated with improved cardiomyocyte contractility and protection against apoptosis.