Thermo-responsive nanocarriers aim to improve the delivery of drugs into target tissue by a process of size-mediated deposition activated by thermal stimuli. The direct imaging of thermally-induced changes in nanocarrier morphology was demonstrated using in situ liquid AFM over a nano-scale and temperature range relevant for clinical approaches. In situ AFM proved to be a unique method for investigating the dynamic conformational changes of individual nanoparticles, promoting its application in the future development of stimuli-responsive nanocarriers.