Microbial metabolomic analysis is essential for understanding responses of microorganisms to heat stress. To understand the comprehensive metabolic responses of Escherichia coli to continuous heat stress, we characterized the metabolomic variations induced by heat stress using NMR spectroscopy in combination with multivariate data analysis. We detected 15 amino acids, 10 nucleotides, 9 aliphatic organic acids, 7 amines, glucose and its derivative glucosylglyceric acid, and methanol in the E. coli extracts. Glucosylglyceric acid was reported for the first time in E. coli. We found that heat stress was an important factor influencing the metabolic state and growth process, mainly via suppressing energy associated metabolism, reducing nucleotide biosynthesis, altering amino acid metabolism and promoting osmotic regulation. Moreover, metabolic perturbation was aggravated during heat stress. However, a sign of recovery to control levels was observed after the removal of heat stress. These findings enhanced our understanding of the metabolic responses of E. coli to heat stress and demonstrated the effectiveness of the NMR-based metabolomics approach to study such a complex system.