Extracellular vesicles (EVs) play an important role in intercellular communication and are involved in both physiological and pathological processes. In the central nervous system (CNS), EVs secreted from different brain cell types exert a sundry of functions, from modulation of astrocytic proliferation and microglial activation to neuronal protection and regeneration. However, the effect of aging on the biological functions of neural EVs is poorly understood. In this work, we studied the biological effects of small EVs (sEVs) isolated from neural cells maintained for 14 or 21 days in vitro (DIV). We found that EVs isolated from 14 DIV cultures reduced the extracellular levels of lactate dehydrogenase (LDH), the expression levels of the astrocytic protein GFAP, and the complexity of astrocyte architecture suggesting a role in lowering the reactivity of astrocytes, while EVs produced by 21 DIV cells did not show any of the above effects. These results in an in vitro model pave the way to evaluate whether similar results occur in vivo and through what mechanisms.