Metabolic syndrome, featured by obesity and diabetes, is an independent risk factor for cardiovascular disease. The high availability of free fatty acids characteristic of obesity has been proposed to increase fatty acid oxidation, impair mitochondrial function, stimulate reactive oxygen species (ROS) production and eventually lead to oxidative stress, which then would cause myocyte damage and heart dysfunction. However, the role of oxidative stress as the initial cause of obesity-induced heart failure is controversial. We evaluated the time-dependent effect of fatty acids in vitro in adult rat cardiac myocytes (treated with palmitate and oleate, the major saturated and unsaturated fatty acids in the plasma, respectively), and in vivo in mice fed a high-fat diet (fat calories = 60%). Palmitate and oleate dose-dependently stimulated mitochondrial respiration and superoxide flash activity, a respiration-coupled bursting ROS production event in single mitochondria. The flash frequency increased 2-fold (0.1 mM, the physiological level in plasma) and 3-fold (0.3 mM, the plasma level found in obese subjects) after only 2 hr incubation and up to 24 hr, increase that could be blocked by the CPT1 inhibitor etomoxir (100uM). Surprisingly, there was no obvious mitochondrial oxidative stress, although the high level of fatty acids caused mPTP opening and cell death at 48 hr. Further, aconitase activity was normal and NAD/NADH ratio was decreased, indicating a more reduced status rather than oxidative stress. Intriguingly, mitochondrial membrane potential was slightly lowered after only 2 hr of treatment. Finally, the high-fat diet-fed mice developed heart dysfunction after 12 weeks of treatment, but oxidative stress only appeared after 18 weeks. In the light of these results, we hypothesize that high fatty acid supply induced multiple adaptive and mal-adaptive responses in cardiac myocytes. The increased mitochondrial respiration and bursting ROS production is offset by a mild uncoupling and increased reducing equivalents. Therefore, oxidative stress is unlikely an initial cause of myocyte death and heart dysfunction in obesity, but rather a consequence of mitochondrial dysfunction, although the exact cause of this dysfunction remains to be elucidated.