Trichinella spiralis muscle larvae-derived extracellular vesicles (TsEV) are key mediators of host–parasite interaction, yet the impact of isolation protocols on their properties and function remains unclear. This study compared four TsEV isolation protocols differing in culture duration (20 h vs. 4 days) and medium composition (with or without fetal bovine serum, FBS). Dynamic light scattering revealed that FBS supplementation yielded small particles (average ~150 nm), whereas FBS-free protocols produced larger, more heterogeneous populations, particularly with extended culture. Proteomic analysis identified a constitutive protein core across all conditions, alongside protocol-specific proteins, with exportin-1 consistently more abundant under FBS-free conditions irrespective of culture duration. Functional assays in RAW 264.7 macrophages showed that TsEV induced both nitric oxide (NO) and arginase activity, but the magnitude and kinetics of these responses varied markedly depending on the protocol. Among the conditions tested, Protocol 3 (4 days without FBS) induced the highest NO levels, whereas Protocol 4 (4 days with FBS) showed the strongest potentiation of lipopolysaccharide (LPS)-induced arginase. These divergent profiles likely reflect differences in EVs’ size and cargo arising from distinct culture conditions, although the relative contribution of duration and medium composition could not be fully dissected. Our findings demonstrate that culture parameters profoundly influence TsEVs’ properties and immunomodulatory capacity, underscoring the need for standardized isolation protocols.