Abstract Our work in the area of mitochondrial function, energy homeostasis and metabolomics has led us to discover a remarkably strong association between adverse cardiometabolic outcomes and tissue/blood levels of acylcarnitine (AC) conjugates. These metabolites derive from acyl-CoA intermediates of fuel catabolism and permit mitochondrial export of excess carbons. For the past decade, our laboratory has remained keenly committed to answering a crucial question: What is this AC signature telling us about the interplay between mitochondria and metabolic disease? The current proposal aims to test the hypothesis that AC accumulation reflects a bottleneck in the fatty acid oxidation (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 laboratory during the previous grant cycle. 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 graded energetic challenge. We have been combining this platform with mass spectrometry-based metabolomics, proteomics and 13C metabolic flux analysis to evaluate mitochondrial remodeling and corresponding changes in respiratory power and efficiency in response to a variety of nutritional and genetic maneuvers. New and exciting findings suggest that AC accumulation reflects a critical thermodynamic vulnerability in the mitochondrial FAO pathway, and thereby serves as a signal of bioenergetic stress, en route to compromised bioenergetics and impending tissue/organ failure. Moreover, our preliminary studies suggest mitochondria resident in untrained skeletal muscles and failing hearts are especially vulnerable to this lipid-induced “traffic jam”; and that ketones are uniquely able to circumvent the roadblock to defend cellular energetics in settings of metabolic stress. Accordingly, we also aim to test the hypothesis that ketone oxidation plays an essential role in permitting the salutary mitochondrial and metabolic adaptations known to occur in response to regimens of intermittent fasting.