This study aimed to clarify the effects of motor fatigue on cortical activation levels and functional connectivity during upper limb resistance training using functional near-infrared spectroscopy (fNIRS). Ten healthy college students participated in a high intensity upper limb resistance training and fNIRS was used to measure the changes of oxyhemoglobin concentration changes (HbO) in bilateral sensorimotor cortex (SMC), premotor cortex (PMC), supplementary motor area (SMA), and dorsolateral prefrontal cortex (DLPFC). The integral value (IV) of blood oxygen signal was calculated as an indicator of cortical activation level and the whole brain correlation analysis was used to calculate cortical functional connectivity. The results showed that as motor fatigue deepened, the activation levels of bilateral DLPFC and PMC in early stage were significantly higher than those in later stage (P<0.05), and the functional connectivity strength of the motor related cortex areas between the hemispheres was significantly reduced, which was manifested by the functional connectivity strength of LSMC-RSMC and LPMC-RSMC showed a significant decrease in middle stage compared with that in early stage (P<0.05) and that the functional connectivity strength of LPMC-RSMC and RSMC-SMA showed a significant decrease in later stage compared with that in early stage (P<0.05). In each stage, the motor related cortex areas maintained high activation levels and the cerebral cortex showed extensive functional connectivity.Clinical Relevance- The clinical relevance of this study is to deepen the understanding of the neural processes related to upper limb resistance training based on motor fatigue, and provide a clinical basis for optimizing resistance training strategies related to motor dysfunction patients with altered brain function under fatigue.