The discovery of novel serological biomarkers is critical for improving disease diagnosis and monitoring treatment response. Proteomic analysis of model systems, such as isolated cells in culture and patient plasma and serum, represents the current state-of-the-art. Here, we coupled proteomics with isolated organ perfusion, which allows a disease state to be studied in a physiologic, yet controlled, environment. Potential markers specific to the disease or to changes in the surrounding tissue may be discovered. The effectiveness of this model was evaluated using proteomic analysis of effluent fractions collected from isolated beating rat hearts during reperfusion after brief episodes of ischemia. The detection of clinical markers for myocardial ischemia in this effluent was robust and analytically straightforward, validating the potential of isolated organ perfusion in diagnostic protein discovery.