Fish oil (FO) and tetradecylthioacetic acid (TTA) - a synthetic modified fatty acid have beneficial effects in regulating lipid metabolism. In order to dissect the mechanisms underlying the molecular action of those two fatty acids we have investigated the changes in mitochondrial protein expression in a long-term study (50weeks) in male Wistar rats fed 5 different diets. The diets were as follows: low fat diet; high fat diet; and three diets that combined high fat diet with fish oil, TTA or combination of those two as food supplements. We used two different proteomics techniques: a protein centric based on 2D gel electrophoresis and mass spectrometry, and LC-MSE based peptide centric approach. As a result we provide evidence that fish oil and TTA modulate mitochondrial metabolism in a synergistic manner yet the effects of TTA are much more dramatic. We demonstrate that fatty acid metabolism; lipid oxidation, amino acid metabolism and oxidative phosphorylation pathways are involved in fish oil and TTA action. Evidence for the involvement of PPAR mediated signalling is provided. Additionally we postulate that down regulation of components of complexes I and II contributes to the strong antioxidant properties of TTA. BIOLOGICAL SIGNIFICANCE: This study for the first time explores the effect of fish oil and TTA - tetradecyl-thioacetic acid and the combination of those two as diet supplements on mitochondria metabolism in a comprehensive and systematic manner. We show that fish oil and TTA modulate mitochondrial metabolism in a synergistic manner yet the effects of TTA are much more dramatic. We demonstrate in a large scale that fatty acid metabolism and lipid oxidation are affected by fish oil and TTA, a phenomenon already known from more directed molecular biology studies. Our approach, however, shows additionally that amino acid metabolism and oxidative phosphorylation pathways are also strongly affected by TTA and also to some extent by fish oil administration. Strong evidence for the involvement of PPAR mediated signalling is provided linking the different metabolic effects. The global and systematic viewpoint of this study compiles many of the known phenomena related to the effects of fish oil and fatty acids giving a solid foundation for further exploratory and more directed studies of the mechanisms behind the beneficial and detrimental effects of fish oil and TTA diet supplementation. This work is already a second article in a series of studies conducted using this model of dietary intervention. In the previous study (Vigerust et al., ) the effects of fish oil and TTA on the plasma lipids and cholesterol levels as well as key metabolic enzymes in the liver have been studied. In an ongoing study more work is being done to explore in detail for example the link between the down regulation of the components of the respiratory chain (observed in this study) and the strong antioxidant effects of TTA. The reference diet in this study has been designed to mimic an unhealthy - high fat diet that is thought to contribute to the development of metabolic syndrome - a condition that is strongly associated with diabetes, obesity and heart failure. Fish oil and TTA are known to have beneficial effects for the fatty acid metabolism and have been shown to alleviate some of the symptoms of the metabolic syndrome. To date very little is known about the molecular mechanisms behind these beneficial effects and the potential pitfalls of the consumption of those two compounds. Only studies of each compound separately and using only small scale molecular biology approaches have been carried out. The results of this work provide an excellent starting point for further studies that will help to understand the metabolic effects of fish oil and TTA and will hopefully help to design dietary programs directed towards reduction of the prevalence of metabolic syndrome and associated diseases.