High temperature is a powerful stressor for fish living in natural and artificial environments, especially for cold water species. Understanding the impact of thermal stress on physiological processes of fish is crucial for better cultivation and fisheries management. However, the metabolic mechanism of cold water fish to thermal stress is still not completely clear. In this study, a NMR-based metabonomic strategy in combination with high throughput RNA-Seq was employed to investigate global metabolic changes of plasma and liver in a typical cold water fish species lenok (Brachymystax lenok) subjected to a sub-lethal high temperature. Our results showed that thermal stress caused multiple dynamic metabolic alterations of the lenok with prolonged stress, including repression of energy metabolism, shifts in lipid metabolism, alterations in amino acid metabolism, changes in choline and nucleotide metabolisms. Specifically, thermal stress induced an activation of glutamate metabolism, indicating that glutamate could be an important biomarker associated with thermal stress. Evidence from Hsp 70 gene expression, blood biochemistry and histology confirmed that high temperature exposure had negative effects on health of the lenok. These findings imply that thermal stress has a severe adverse effect on fish health and demonstrate that the integrated analyses combining NMR-based metabonomics and transcriptome strategy is a powerful approach to enhance our understanding of metabolic mechanisms of fish to thermal stress.