Synthetic lipid vesicles are valuable mesoscale molecular confinement vessels for studying membrane mechanics and lipid-protein interactions, and they have found vast utility among bio-inspired technologies including drug delivery vehicles. Having a diameter of a few tens to hundreds of nanometers enables such complex processes to be studied at the level of a handful of molecules, conferring benefits in exploring molecular heterogeneity under near-physiological conditions. Vesicle morphology can be modified by changing the mixture of lipids used in creating them, fusing and lysing vesicles using proteins and detergents, or modifying the pH, temperature and ionic strength of the surrounding buffer, enabling the effects of membrane curvature on these processes to be explored. The requirements for experimental control have meant that most vesicle studies are performed under dilute solution conditions in vitro. The use of vesicles in crowded intracellular environments, known to influence the activities of many cellular processes, is limited by our knowledge of how molecular crowders affect vesicle morphology. To tackle this limitation, we used fluorescence spectroscopy, picosecond time correlated single photon counting and single-vesicle imaging to explore the influence of molecular crowding on the structure of freely-diffusing and surface-tethered vesicles fluorescently labelled with DiI and DiD. By quantifying single-molecule Förster resonance energy transfer (smFRET) between the probes, we determined the dependence on vesicle morphology from crowding using the molecular weight crowders sorbitol PEG400, Ficoll400 and PEG8000, identifying a common theme that both low and high molecular weight crowders trigger structural rearrangements of the vesicle that we assign to compaction. A particularly striking observation is that the low molecular weight crowder sorbitol results in irreversible changes to the smFRET efficiency attributed to permanent compaction, whereas the influence of the higher molecular weight crowders was found to be reversible. The effect of crowding perturbation on the architecture of such a reduced system not only emphasizes the power of single-vesicle approaches to probe complex biology, but also illustrates the potential to controllably alter the vesicle volume and radius of curvature for several biotechnological applications.