<p>Locally advanced rectal cancer is treated with neoadjuvant-chemoradiotherapy, however only 22% of patients achieve a complete response. Resistance mechanisms are poorly understood. Radiation-induced Bystander Effect (RIBE) describes the effect of radiation on neighbouring unirradiated cells. We investigated the effects of ex vivo RIBEinduction from normal and rectal cancer tissue on bystander cell metabolism, mitochondrial function and metabolomic profiling. We correlated bystander events to patient clinical characteristics. Ex vivo RIBE-induction caused metabolic alterations in bystander cells, specifically reductions in OXPHOS following RIBE-induction in normal (p = 0.01) and cancer tissue (<i>p</i> = 0.03) and reduced glycolysis following RIBE-induction in cancer tissue (<i>p</i> = 0.01). Visceral fat area correlated with glycolysis (<i>p</i> = 0.02) and ATP production (<i>p</i> = 0.03) following exposure of cells to TCM from irradiated cancer biopsies. Leucine levels were reduced in the irradiated cancer compared to the irradiated normal secretome (<i>p</i> = 0.04). ROS levels were higher in cells exposed to the cancer compared to the normal secretome (<i>p</i> = 0.04).</p> <p>RIBE-induction ex vivo causes alterations in the metabolome in normal and malignant rectal tissue along with metabolic alterations in bystander cellular metabolism. This may offer greater understanding of the effects of RIBE on metabolism, mitochondrial function and the secreted metabolome.</p>