Plant-derived extracellular vesicles possess different intrinsic therapeutic activities that can potentially help prevent or treat human diseases. These EVs can be administered as such, or ingested daily in the diet. However, food EV sources are commonly subjected to cooking processes prior to consumption. Indeed, before any biological effect can be exerted, orally-administered EVs must overcome several technological, extracellular, and cellular barriers before reaching target tissues. To explore their capacity to exert biological effects, we evaluated the stability of broccoli-derived EVs under thermal culinary conditions and gastrointestinal digestion (GI), and their ability to cross the intestinal barrier. EVs were isolated from raw broccoli, broccoli sprouts and broccoli subjected to one of five thermal culinary treatments (frying, boiling, microwaving, scalding and steaming). Nanoparticle tracking analysis (particle size distribution, concentration, and z-potential), electron microscopy, and Western blot were used to characterize the EVs. They were run through a simulated in vitro GI tract and again characterized. Potential EV transport through the intestinal barrier was assessed using transwell-cultured differentiated Caco-2 cells, and their antiproliferative effects were evaluated in intestinal and hepatic cell lines. All five thermal culinary treatments lowered EV particle concentration, protein content, and cargo, as well as influencing EV structure. Around 10 % of the broccoli-derived EVs survived GI digestion. The RNA cargo of EVs can enter intestinal cells, but only a small amount of EVs cross the intestinal barrier. Thermal treatment also reduced EV antiproliferative effects. Broccoli-derived EVs are affected by thermal culinary processing and GI digestion, and cannot cross the intestinal barrier. However, even after thermal processing they apparently maintain some potential therapeutic effects. © 2025 Elsevier B.V., All rights reserved.