Liposomes required for drug delivery are commonly obtained by extrusion of phospholipid vesicle suspensions through track-etched membranes. The effects of trans-membrane pressure, membrane pore size and bilayer composition on extruded liposome size are well-studied. Vesicle suspensions used in these extrusion studies are highly polydisperse, ranging from 20 nm to 100 μm. Vesicle sub-populations smaller than membrane pore size do not undergo extrusion-mediated size reduction and contribute significantly to the mean radius of extruded liposomes. In the present work, giant unilamellar vesicles (GUVs) are isolated by subjecting electroformed vesicle suspensions to low-pressure filtration. The isolated GUVs are extruded through track-etched polycarbonate membranes with pore radii ranging from 25 to 200 nm. We show that, when vesicles larger than the membrane pore size are extruded, the minimum attainable value of mean radius of resulting liposomes is independent of initial vesicle size as well as the number of extrusion cycles. We also show that bilayer composition significantly influences the extruded liposome size. These results provide new insights into the possible mechanisms of vesicle size reduction during extrusion process.