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Substrate utilization in tissues with high energetic requirements could play an important role in cardiometabolic disease. Current techniques to assess energetics are limited by high cost, low throughput, and the inability to resolve multiple readouts simultaneously. Consequently, we aimed to develop a multiplexed optical imaging platform to simultaneously assess energetics in multiple organs in a high throughput fashion.The detection of 18F-Fluordeoxyglucose uptake via Cerenkov luminescence and free fatty acid uptake with a fluorescent C16 free fatty acid was tested. Simultaneous uptake of these agents was measured in the myocardium, brown/white adipose tissue, and skeletal muscle in mice with/without thoracic aortic banding. Within 5 weeks of thoracic aortic banding, mice developed left ventricular hypertrophy and brown adipose tissue activation with upregulation of β3AR (β3 adrenergic receptors) and increased natriuretic peptide receptor ratio. Imaging of brown adipose tissue 15 weeks post thoracic aortic banding revealed an increase in glucose (P<0.01) and free fatty acid (P<0.001) uptake versus controls and an increase in uncoupling protein-1 (P<0.01). Similar but less robust changes were seen in skeletal muscle, while substrate uptake in white adipose tissue remained unchanged. Myocardial glucose uptake was increased post-thoracic aortic banding but free fatty acid uptake trended to decrease.A multiplexed optical imaging technique is presented that allows substrate uptake to be simultaneously quantified in multiple tissues in a high throughput manner. The activation of brown adipose tissue occurs early in the onset of left ventricular hypertrophy, which produces tissue-specific changes in substrate uptake that may play a role in the systemic response to cardiac pressure overload.