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Description: Fatigue perhaps is the most common complaint in clinic, while it is also one of the least understood phenomena in neuroscience. With the development of in vivo glutamate measurement technique, the role of glutamate in the pathophysiology of fatigue has emerged. Compared to the conventional 1H-MRS, glutamate chemical exchange saturation transfer (GluCEST) MRI is a recently developed imaging technology with high sensitivity and special resolution, which has been used in more and more fields. However, it has not been applied to measure glutamate levels in fatigue. In the present study we employed GluCEST technique to measure brain glutamate levels in an animal model of prolonged fatigue. We found a significant increase of glutamate level in the whole brain. A further analysis of different sub-regions of brain relevant to pathophysiology of fatigue including the hippocampus, striatum and prefrontal cortex revealed a trend of increase, although no statistical significance was reached. Our study indicates that the increment of brain glutamate may be part of the pathophysiological process of fatigue. Our work may inspire interest in the study of the relationship between fatigue and glutamate, which may be crucial for understanding fatigue, a confusing biological phenomena worthy of more attention and investigation.