Excess lipid accumulation resulting from an elevated supply of plasma fatty acids is linked to the pathogenesis of the metabolic syndrome and heart disease. The term lipotoxicity was coined to describe how lipid accumulation leads to cellular dysfunction and death, and these events are demonstrated in non-adipose tissues including heart, pancreas and liver. While lipotoxicity was reported in cultured skeletal muscle cells, the degree of lipotoxicity in vivo and the functional consequences is unresolved. We studied three models of fatty acid overload in male mice: 5 h Intralipid(R) and heparin infusion, prolonged high fat feeding (HFF) and genetic obesity induced by leptin deficiency (ob/ob mice). Markers of apoptosis, proteolysis and autophagy were assessed as readouts of lipotoxicity. The lipid infusion increased caspase 3 activity in skeletal muscle, demonstrating that enhancing fatty acid flux activates pro-apoptotic pathways in vivo. HFF and genetic obesity increased tissue lipid content but did not influence apoptosis. Gene array analysis revealed that HFF reduced the expression of 31 pro-apoptotic genes. Markers of autophagy (LC3beta and beclin-1 expression) were unaffected by HFF and were associated with enhanced Bcl2 protein expression. Proteolytic activity was similarly unaffected by HFF or in ob/ob, mice. Thus, contrary to our previous findings in muscle culture in vitro and in other non-adipose tissues in vivo, fatty acid overload did not induce apoptosis, autophagy or proteolysis in skeletal muscle. A broad transcriptional suppression of pro-apoptotic proteins may explain this resistance to lipid-induced cell death in skeletal muscle in vivo.