It is well known that few weeks of high fat (HF) diet may induce metabolic disturbances and mitochondrial dysfunction in skeletal muscle. However, little is known about the effects of long-term HF exposure and effects on brain mitochondria are unknown. Wistar rats were fed either chow (13E% fat) or HF diet (60E% fat) for 1 year. The HF animals developed obesity, dyslipidemia, insulin resistance, and dysfunction of isolated skeletal muscle mitochondria: state 3 and state 4 were 30% to 50% increased (P<0.058) with palmitoyl carnitine (PC), while there was no effect with pyruvate as substrate. Adding also succinate in state 3 resulted in a higher substrate control ratio (SCR) with PC, but a lower SCR with pyruvate (P<0.05). The P/O2 ratio was lower with PC (P<0.004). However, similar tests on isolated brain mitochondria from the same animal showed no changes with the substrates relevant for brain (pyruvate and 3-hydroxybutyrate). Thus, long-term HF diet was associated with obesity, dyslipidemia, insulin resistance, and significantly altered mitochondrial function in skeletal muscle. Yet, brain mitochondria were unaffected. We suggest that the relative isolation of the brain due to the blood-brain barrier may play a role in this strikingly different phenotype of mitochondria from the two tissues of the same animal.