Chitooligosaccharides (COS), oligosaccharides composed of two to seven glucosamine residues, are known to exhibit various biological activities. In this study, we investigated the effects of COS in an in vivo mouse sleep deprivation-induced fatigue model in an effort to develop a functional food with anti-fatigue efficacy. Male Balb/c mice were orally administered 500 mg (kg d)⁻¹ of COS lactate or COS HCl for 2 weeks, and severe fatigue was induced by sleep deprivation. To evaluate the extent of fatigue, the swimming time, representing the immobility time, was measured in a forced swim test. As a result, oral intake of COS lactate-manifested anti-fatigue effects could be observed by the attenuation of fatigue-induced body weight loss and shorter immobility period. In addition, COS lactate was shown to alleviate the fatigue-induced increase in cortisol and lipid peroxidation and a decrease in superoxide dismutase (SOD) activity. Of particular note, the oral administration of COS lactate increased the mitochondrial membrane potential and the mitochondrial number significantly, indicating that COS lactate may enhance mitochondrial function. In support of this, COS lactate increased the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and cytochrome c (Cyt C) mRNA, indicating that it may increase mitochondrial biogenesis. These results suggest that COS lactate can be an effective anti-fatigue functional food, and this anti-fatigue effect may result from, at least in part, the enhancement of mitochondrial biogenesis and the inhibition of free radical generation.