Electrolysed water (EW) is an activated liquid with a high oxidation-reduction potential. EW causes oxidative damage to pathogenic microorganisms and as a result, may have utility in the food industry. The molecular mechanism of EW's action is not understood. In this study, we exposed Escherichia coli ATCC 25922 to a sub-lethal concentration of EW and examined structural and metabolic changes. Atomic force microscopy revealed that EW caused damage to E. coli membranes. To understand the metabolic responses to EW perturbations in of E. coli, multivariate data analysis of NMR spectroscopy demonstrated that EW significantly influenced the metabolic state. This included reducing nucleotide and amino acid biosynthesis, suppressing energy-associated metabolism, altering osmotic adjustment, and promoting fatty acid metabolism. The results enrich our understanding of E. coli metabolic changes caused by EW perturbation and support the effectiveness of the NMR metabolomics as a valuable tool to analyse and evaluate such a complex biological system.