Uncoupling proteins (UCP) translocate protons from the mitochondrial intermembrane space to the matrix, thereby "uncoupling" electron transport from the production of ATP. It has been shown that these proteins are highly expressed in animals maintained on the ketogenic diet (KD). Although the exact mechanism remains unclear, it is known that these proteins are activated within a protective antireactive oxygen species (ROS) mechanism by free fatty acids (FFA). In our current studies, mitochondrial samples were probed for the presence of UCP2, which is the most ubiquitously expressed UCP isoform. We found that both traumatic brain injury and fasting upregulated the expression of UCP2, with a synergistic upregulation in fasted injured animals. We then used mitochondria from fasted naive animals to screen a number of FFA for their activation of uncoupling as well as their ability to reduce ROS. We found that arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), palmitoleic acid, myristic acid, and butyric acid increased mitochondrial uncoupling when added after oligomycin. These FFA, along with oleic acid, also reduced ROS in mitochondria incubated with oligomycin. In order to correlate our data to KD and fasting, both of which have been shown to be neuroprotective after neurologic insult, we determined the serum levels of FFA in KD and fasted animals using gas chromatography/mass spectroscopy. We also determined brain and cerebrospinal fluid (CSF) FFA levels from fasted animals.