The advent of high-speed atomic force microscopy (HS-AFM) has opened a novel research field for the dynamic analysis of single unlabeled bio-molecules. The integration of a non-disturbing buffer exchange system, allowing the gradual change of the environmental conditions during HS-AFM operation, provides a breakthrough towards the performance of structural titration experiments. Analyzing membrane proteins in membranes and under controlled exposure to environmental stimuli, we report conformational changes of a transporter in response to substrates, of receptors and of ion channels upon ligand binding. Altogether, we showcase the unique potential of HS-AFM, providing ~1nm lateral, ~0.1nm vertical and ~100ms temporal resolution, for visualizing the dynamics of membrane proteins, label-free and in close-to-native conditions.