Analysis of structural and functional parameters of HDL has gained significant momentum in recent years because they are stronger predictors of cardiovascular risk than HDL-cholesterol levels. Surprisingly, in most HDL studies, very low attention is paid to HDL storage, which might critically affect functional properties. In the present study, we systematically examined the impact of storage and freezing on the structural/functional properties of freshly isolated HDL. Initial damage to HDL starts between week 1 and week 4 of storage. We observed that prolonged freezing at −20°C or −70°C led to a shedding of apoA-I from HDL and to the formation of large protein-poor particles, indicating that HDL is irreversibly disrupted. These structural alterations profoundly affected key metrics of HDL function, including HDL-cholesterol efflux capacity and HDL paraoxonase activity. Flash-freezing of isolated HDL prior to storage at −70°C did not preserve HDL structure. However, addition of the cryoprotectants, sucrose or glycerol, completely preserved structure and function of HDL when stored for at least 2 years. Our data clearly indicate that HDL is a complex particle requiring special attention when stored. Addition of cryoprotectants to isolated HDL samples before storage will make biochemical and clinical HDL research studies more reproducible and comparable.