Purpose Retrieving lungs from Category 1 non-heart-beating donors (NHBDs) for transplant (LTX) requires a mandatory period of ischemia. NHBDs can be ventilated before retrieval. In earlier studies, O2-vent of a NHBD delayed cell death, and improved function after LTX. 1 H Nuclear Magnetic Resonance Spectroscopy (NMR-spec) identifies all metabolites in an organ. We characterized lung metabolic phenotype with NMR-spec. Methods and Materials To simulate failed resuscitation in the field (Category 1 NHBD), 6 rats were anesthetized, ventilated briefly with 100% O2 via tracheotomy, sacrificed, then the trachea was clamped. After rapid sternotomy, the right upper lobe was ligated, excised, and flash frozen. Inflated but non-vent lobes from 3 rats were ligated and excised hourly x 4. 3 rats were ventilated 1 hr after death with 100% O2 for 1 hr to simulate O2-vent of a NHBD after consent for lung retrieval. NMR spectra were analyzed with Principal Component Analysis and Orthogonal Partial Least Squares Discriminant Analysis. Results Of nearly 50 metabolites detected by 1 H NMR spec, changes were observed in 23 over 4 hr. Glycolysis persists, more in non-vent lungs. Krebs cycle activity is higher in vent lungs, perhaps due to motion redistributing metabolites. Conclusions NMR-spec can define changes in the lung metabolome due to ischemia. O2-vent of a NHBD started 1 hr after death changes the lung metabolome resulting in more Krebs cycle activity. NMR-spec may identify biomarkers of function, or therapeutic targets to administer to NHBDs. Metabolites Pathways Metabolites Ventilated Non-Vent Krebs (TCA) Cycle Succinate, Glutamine ↔ Krebs (TCA) Cycle Glutamate ↔ ↓ Glycolysis Glucose ↓ ↓ Glycolysis Lactate, Alanine β-Oxidation/Ketone Bodies Acetate, β-hydroxybutyrate ↓ ↓ Phospholipid Intermediates Choline, Phosphocholine, Glycerophsophocholine Nucleotide energy ATP/ADP ↓ ↓ Nucleotide degradation AMP, Inosine, Xanthine, Hypoxanthine Osmolytes/GSH degradation Taurine Amine acceptor Arginine ↔ Amino Acids Leucine, Valine, Isoleucine, Glycine