The dynamic nature of the peptide based lipid bilayer nanodiscs were successfully monitored using high speed atomic force microscopy (HS-AFM) experiments for the first time. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids were used to make nanodiscs of different size by adding the peptide to lipid in the ratio of 1:1 or 1:5 w/w. The SEC profiles of nanodiscs made with 4F:DMPC 1:1 and 1:5 w/w showed an elution profile peak maximum at 10.7 and 13.5 mL, respectively. These elution profiles suggest the formation of nanodiscs with their size varying with the peptide:lipid ratio. The resulting nanodiscs were also characterized by DLS experiments, which showed a hydrodynamic diameter of 12 nm for 1:5 w/w and 6 nm for 1:1 w/w 4F: DMPC. The nanodiscs were further characterized using AFM experiments, which revealed a distinct size difference between the nanodiscs formed with two different peptide: lipid ratios.The real-time monitoring of nanodiscs over time in unprecedented time and space resolutions provides evidence for nanodiscs association and the time scale of associations. The lipid exchange between the nanodiscs monitored using 31P NMR experiments suggest that the exchange of membrane components between nanodiscs that could alter a membrane protein’s structure, dynamics and function can also be characterized at high resolution.