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Phytochemicals have multifunctional bioactivities, such as anti-inflammatory and anti-obesity activities. However, there are still relatively few studies that have investigated the possible anti-fatigue of functional phytochemical compounds (such as resveratrol, triterpenoids, and catechins) in animal models. Therefore, the aim of this study was to develop of in vitro models as tools for cellular and molecular studies of anti-fatigue. C2C12 myotubes were used for evaluating in vitro the anti-fatigue of functional compounds. The results indicated that treatment with some functional compounds (including resveratrol, quercetin, epigallocatechin gallate, and epicatechin) significantly increased the expression of mitochondrial DNA genes (including cytochrome b and cytochrome c oxidase subunit II ) and the activities of mitochondrial enzymes (including citrate synthase and cytochrome c oxidase) in C2C12 myotubes. These results demonstrated that resveratrol, quercetin, epigallocatechin gallate, and epicatechin have great potential as novel mitochondrial-activating agents that may play an important role in anti-fatigue.