Nanographene oxide (nGO)-mediated hyperthermia has been increasingly investigated as a localised, minimally invasive anticancer therapeutic approach. Near InfraRed (NIR) light irradiation for inducing hyperthermia is particularly attractive, because biological systems mostly lack chromophores that absorb in this spectral window, facilitating the selective heating and destruction of cells which have internalized the NIR absorbing-nanomaterials. However, little is known about biological effects accompanying nGO-mediated hyperthermiaat cellular and molecular levels.In this work, well-characterised pegylatednGOsheets with an hydrodynamic size of 300 nm were incubated with human Saos-2 osteosarcoma cells for 24h and their incorporation verified by flow cytometry and confocal microscopy. No effect on cell viability was observed after nGO incorporation by Saos-2 cells. However, a proliferation delay was observed due to the presence of nGO sheets in the cytoplasm.1H NMR metabolomicswas employed to screen for changes in the metabolic profile of cells, as this could help to improve understanding of celular responses to nanomaterials and provide new endpoint markers of effect.Cells incorporating nGO sheets showed noticeable changes in 10 metabolites compared to control cells, including decreased levels of several amino acids, taurine and creatine and increased levels of phosphocholine and uridine/adenosine nucleotides. After NIR irradiation, cells showed decreases in glutamate and uridine nulceotides, together with increases in glycerophosphocholine and adenosine monophosphate. Overall, this study has shown that the celular metabolome sensitively responded to nGO exposure and nGO-mediated hyperthermia and that NMR metabolomics is a powerful tool toinvestigate treatment responses.