Essentials The metabolic integration processes required for platelet activation are unclear. The metabolic plasticity of human platelets were investigated. Activated platelets exhibit a glycolytic phenotype while preserving mitochondrial function. Platelets can switch freely between glucose/glycogen and fatty acids to support aggregation. SUMMARY: Background Platelet activation is an energy-dependent process, but the type and integrated use of metabolic fuels required to drive activation remain unclear. Objective To dissect the metabolic fuel and pathway plasticity required for platelet activation. Methods Platelet oxygen consumption rate and extracellular acidification rate were measured as markers of oxidative phosphorylation (OXPHOS) and glycolysis, respectively. Glucose and glycogen were quantified by enzyme-coupled fluorometric assay. Results Blood platelets switched freely between glycolysis and OXPHOS, using either glucose or fatty acids at rest. The transition of platelets from a quiescent to an activated state promoted rapid uptake of exogenous glucose, associated with a shift to a predominantly glycolytic phenotype coupled with a minor rise in mitochondrial oxygen consumption. Consistent with this metabolic plasticity, under nutrient-limiting conditions, platelets utilized glucose, glycogen or fatty acids independently to support activation. Importantly, the glycolytic switch occurred even in the absence of extracellular glucose, originating from endogenous glycogen. Focusing on the relative flexibility of mitochondrial fuel oxidation of glucose and fatty acids, we found that inhibition of oxidation of a single fuel was compensated for by increased oxidation of the other, but, when oxidation was inhibited, glycolysis was upregulated. Glutamine made little contribution to mitochondrial oxygen consumption. Analysis of platelet functional dependency on ATP from different pathways demonstrated that inhibition of both fuel oxidation and glycolysis were required to prevent agonist-driven platelet activation. Conclusion Platelets have significant metabolic fuel and pathway flexibility, but preferentially use glycolysis for ATP generation when activated.