Persons with Down syndrome (DS) exhibit low muscle strength that significantly impairs their physical functioning. The Ts65Dn mouse model of DS also exhibits muscle weakness in vivo and may be a useful model to examine DS-associated muscle dysfunction. Therefore, the purpose of this experiment was to directly assess skeletal muscle function in the Ts65Dn mouse and to reveal potential mechanisms of DS-associated muscle weakness. Soleus muscles were harvested from anesthetized male Ts65Dn and wild-type (WT) colony controls. In vitro muscle contractile experiments revealed normal force generation of nonfatigued Ts65Dn soleus, but a 12% reduction in force was observed during recovery from fatiguing contractions compared with WT muscle (P < 0.05). Indicators of oxidative stress and mitochondrial oxidative capacity were assessed to reveal potential mechanisms of DS-associated muscle weakness. Protein expression of copper-zinc superoxide dismutase (SOD1), a triplicated gene in persons with DS and Ts65Dn mice, was increased 25% (P < 0.05) in Ts65Dn soleus. Nontriplicated antioxidant protein expression was similar between groups. Lipid peroxidation was unaltered in Ts65Dn animals, but protein oxidation was 20% greater compared with controls (P < 0.05). Cytochrome-c oxidase expression was 22% lower in Ts65Dn muscle (P < 0.05), while expression of citrate synthase was similar between groups. Microarray analysis revealed alteration of numerous pathways in Ts65Dn muscle, including proteolysis, glucose and fat metabolism, neuromuscular transmission, and ATP biosynthesis. In summary, despite biochemical and gene expression differences in soleus muscle of Ts65Dn animals, the functional properties of skeletal muscle likely contribute a minor part to the in vivo muscle weakness.