Abstract Our work in the areas of mitochondrial function, energy homeostasis, and metabolomics has led us to discover a remarkably strong association between adverse cardiometabolic outcomes and elevated blood/tissue levels of acylcarnitine (AC) conjugates. These metabolites derive from acyl-CoA intermediates of fuel catabolism and permit mitochondrial export of excess carbons. Most ACs are generated as byproducts in incomplete fatty acid oxidation (FAO). For the past fifteen years, our investigative team has remained keenly committed to answering a crucial question: What is this AC signature telling us about the interplay between mitochondrial fitness and metabolic resilience? Our recent preclinical studies lead us to hypothesize that AC accumulation reflects a bottleneck in the FAO pathway that diminishes mitochondrial power and efficiency. This prediction stems from unique insights gained via the application of a new mitochondrial diagnostics platform developed by our team. In simple terms, our assays serve as an in vitro “stress test” that evaluates how well a given population of mitochondria, fueled by specific mixtures of carbon substrates, responds to a lipid challenge. These studies revealed that “mitochondrial lipid tolerance” predicts tissue and whole-body metabolic health. Moreover, by combining this platform with mass spectrometry-based metabolomics and proteomics, we uncovered a critical vulnerability in the mitochondrial FAO pathway that associates with “mitochondrial lipid intolerance” and whole- body metabolic dysfunction. We also found that mitochondrial “lipid tolerance”–defined as the ability of mitochondrial to maintain bioenergetic stability in the face of a lipid challenge–strongly correlates with the activity of a short chain carbon circuit that enables reverse flux of acetyl CoA through medium-chain ketothiolase (MKT) to regenerate free CoA and oxidized NAD+, both critical co-factors necessary for oxidative metabolism. We also discovered that reverse SC carbon flux through this circuit depends on an ample supply of acetyl CoA derived from either pyruvate (in the fed state) or ketones (in the fasted state). Together, these findings lead us to hypothesize that reverse flux through the SC carbon circuit, supported by pyruvate and/or ketones, plays a crucial role in conferring whole body metabolic fitness and metabolic resilience. The research plan developed for the next cycle of this grant aims to determine whether reverse flux through the SC carbon circuit is both necessary and sufficient to confer mitochondria lipid tolerance, metabolic flexibility, and metabolic adaptations to exercise training.