PURPOSECardiac metabolism is altered in heart failure and ischemia-reperfusion injury states. We hypothesized that metabolomic profiling during ex-situ normothermic perfusion before heart transplantation (HT) would lend insight into myocardial substrate utilization and report on subclinical and clinical allograft dysfunction risk. METHODSMetabolomic profiling was performed on serial samples of ex-situ normothermic perfusate assaying biomarkers of myocardial injury in lactate and cardiac troponin I (TnI) as well as metabolites (66 acylcarnitines, 15 amino acids, non-esterified fatty acids (NEFA), ketones, and 3-hydroxybutyrate). We tested for change over time in injury biomarkers and metabolites, along with differential changes by recovery strategy (donation after circulatory death (DCD) v. donation after brain death (DBD)). We examined associations between metabolites, injury biomarkers, and primary graft dysfunction (PGD). Analyses were performed using linear mixed models adjusted for recovery strategy, assay batch, donor predicted heart mass, and time. RESULTSA total of 176 samples from 92 ex-situ perfusion runs were taken from donors with a mean age of 35 years (SD 11.3) years and a median total ex-situ perfusion time of 234 (IQR 84) minutes. Lactate trends over time differed significantly by recovery strategy, while TnI increased during ex-situ perfusion regardless of DCD v. DBD status. We found fuel substrates were rapidly depleted during ex-situ perfusion, most notably the branched-chain amino acids leucine/isoleucine, as well as ketones, 3-hydroxybutyrate, and NEFA (least squares (LS) mean difference from the first to last timepoint -1.7 - -4.5, FDR q<0.001). Several long-chain acylcarnitines (LCAC), including C16, C18, C18:1, C18:2, C18:3, C20:3, and C20:4 increased during the perfusion run (LS mean difference 0.42 – 0.67, q<0.001). Many LCACs were strongly associated with lactate and TnI. The change over time of many LCACs was significantly different for DCD vs. DBD, suggesting differential trends in fuel substrate utilization by ischemic injury pattern. Changes in leucine/isoleucine, arginine, C12:1-OH/C10:1-DC, and C16-OH/C14-DC were associated with increased odds of moderate-severe PGD. Neither end-of-run nor change in lactate or TnI was associated with PGD. CONCLUSIONMetabolomic profiling of ex-situ normothermic perfusion solution reveals a pattern of fuel substrate utilization that correlates with subclinical and clinical allograft dysfunction. This study highlights a potential role for interventions focused on fuel substrate modification in allograft conditioning during ex-situ perfusion to improve allograft outcomes.