Chemical exchange saturation transfer of the relayed nuclear Overhauser effect(rNOE) enables detection of mobile protein protons via their exchange with the water signal. Recent studies have shown a close relationship between the rNOE-signal and the protein conformation. This study used the -3.5ppm rNOE-signal to monitor the heat shock response of HepG2 cells in a microcavity array-based bioreactor system. A significant drop of the rNOE-signal to 92.4±1.3% and a slow, steady increase of the rNOE-signal for 136.1±13.6min after heat shock were observed. Therefore, the rNOE-signal in CEST spectroscopic imaging is a sensitive readout for the cellular heat shock response.