Regulation of gene expression according to the environmental conditions is vitally important for all living organisms. Temperature is one of the key cues for microorganisms that can exist in different environments, and several mechanisms of temperature-dependent re-allocation of cellular resources have been described. Here we compared transcriptome and proteome of E. coli grown at basal (37°C) and febrile temperatures (42°C) of the mammalian host. We demonstrated that the downregulation of Escherichia coli motility at high temperature primarily occurred due to the malfunction of secretion apparatus, apparently related to the disassembly of the flagellar basal body. For proteome analysis we performed data-independent acquisition (DIA) mass spectrometry via Spectronaut. The Spectronaut file can be assessed with the freely available Spectronaut viewer (https://www.biognosys.com/technology/spectronaut-viewer).