The regulation of the phospholipid protrusion, the vertical fluctuation at the surface of cell-sized vesicles (CSVs),was investigated by solution NMR in combination with the 1H–1H nuclear Overhauser effect (NOE) enhancementmeasurement. Using deuterated bisphenol A (BP–d) and palmitic acid (PA–d) as model hydrophobic components,we compared how the lipid protrusion is modulated by the presence of BP–d and PA–d in the fluid, soft CSV mem-brane. The phospholipid protrusion was suppressed by BP–d but not by PA–d, although both BP–d and PA–d werebound to the membrane. The contrasting effect of BP–d and PA–d on the protrusion motion is interpreted by the dif-ference in the mode of interaction between these substances and lipids in the membrane; rather hydrogen bondingthan van der Waals forces is the decisive parameter to regulate the phospholipid protrusion motion.