Ectopic expression of uncoupling protein 1 (UCP1) in skeletal muscle (SM) mitochondria increases lifespan considerably in high-fat diet-fed UCP1 Tg mice compared with wild types (WT). To clarify the underlying mechanisms, we investigated substrate metabolism as well as oxidative stress damage and antioxidant defense in SM of low-fat- and high-fat-fed mice. Tg mice showed an increased protein expression of phosphorylated AMP-activated protein kinase, markers of lipid turnover (p-ACC, FAT/CD36), and an increased SM ex vivo fatty acid oxidation. Surprisingly, UCP1 Tg mice showed elevated lipid peroxidative protein modifications with no changes in glycoxidation or direct protein oxidation. This was paralleled by an induction of catalase and superoxide dismutase activity, an increased redox signaling (MAPK signaling pathway), and increased expression of stress-protective heat shock protein 25. We conclude that increased skeletal muscle mitochondrial uncoupling in vivo does not reduce the oxidative stress status in the muscle cell. Moreover, it increases lipid metabolism and reactive lipid-derived carbonyls. This stress induction in turn increases the endogenous antioxidant defense system and redox signaling. Altogether, our data argue for an adaptive role of reactive species as essential signaling molecules for health and longevity. ; The research leading to these results has received funding from the European Union’s Seventh Framework Program FP7 2007–2013 under grant agreement no. 244995 (BIOCLAIMS Project) and from the German Research Foundation (DFG: KL613/14-2). The studies conducted at the Department of Experimental Medicine were supported in part by research and development grants from the Spanish Ministry of Science and Innovation (BFU2009 – 11879/BFI), the Spanish Ministry of Health (PI08111532), the Autonomous Government of Catalonia (2009SGR735), and COST B35 Action of the European Union.