Exosomes may be used as biomarkers for the prediction and monitoring of response to anti-cancer treatment, yet relevant knowledge is very limited in the case of rectal cancer. Here we applied a combined proteomic and metabolomic approach to reveal exosome components connected with different responses to neoadjuvant radiotherapy in this group of patients and processes associated with identified discriminatory molecules. Moreover, the composition of serum-derived exosomes and a whole plasma was analyzed in parallel to compare the biomarker potential of both specimens, which revealed the highest capacity of exosome proteome to discriminate samples of patients with different responses to radiotherapy.