Ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart in a semi-physiologic state and provides the opportunity to evaluate pre-transplant function. Because of the well-described extraordinary metabolic requirements of the heart, efficient metabolic support is critical for optimal preservation of function and viability. Our aim was to determine changes in energy metabolism/energy substrates during extended ex situ perfusion of hearts in two different perfusion modes, working mode (WM) and non-working mode (NWM).