Abstract <p>The heat shock response is a cellular protection mechanism against sudden temperature upshifts extensively studied in <i>Escherichia coli</i>. However, the effects of thermal evolution on this response remain largely unknown. In this study, we investigated the early and late physiological and transcriptional responses to temperature upshift in a thermotolerant strain under continuous culture conditions. Adaptive laboratory evolution was performed on a metabolically engineered <i>E. coli</i> strain (JU15), designed for <span>d</span>-lactic acid production, to enable cellular growth and fermentation of glucose at 45&#xa0;°C in batch cultures. The resulting homofermentative strain, ECL45, successfully adapted to 45&#xa0;°C in a glucose-mineral medium at pH 7 under non-aerated conditions. The thermal-adapted ECL45 retained the parental strain’s high volumetric productivity and product/substrate yield. Genomic sequencing of ECL45 revealed eight mutations, including one in a non-coding region and six within the coding regions of genes associated with metabolic, transport, and regulatory functions. Transcriptomic analysis comparing the evolved strain with its parental counterpart under early and late temperature upshifts indicated that the adaptation involved a controlled stringent response. This mechanism likely contributes to the strain’s ability to maintain growth capacity at high temperatures.</p> Key points <p>• <i>The temperature upshift response of a thermally adapted strain in continuous culture was studied for the first time.</i></p> <p>• <i>Genomic analyses revealed the presence of a double point mutation in the spoT gene.</i></p> <p>• <i>The thermally adapted strain maintained underexpression of the spoT gene at high temperatures.</i></p> <p>• <i>Supplementation of 0.15 g/L of hydrolyzed protein favored thermal adaptation at 45 °C.</i></p>