Extracellular vesicles (EVs) and their microRNA (miRNA) cargo have been proposed as possible mammary gland health biomarkers in cattle. However, throughout the day, the biologically active milk components, such as miRNAs, may change due to the dynamic nature of milk. The current study aimed to evaluate the circadian fluctuation of milk EVs miRNA cargo to assess the feasibility of milk EVs as future biomarkers for mammary gland health management. Milk from four healthy dairy cows was collected for four consecutive days in the two daily milking sessions in the morning and the evening. The isolated EVs were heterogeneous, intact, and carried the EV protein markers CD9, CD81, and TSG101, as shown by transmission electron microscopy and western blot. The miRNA sequencing results demonstrate that the abundance of miRNA cargo in milk EVs remained stable, unlike other milk components, such as somatic cells, that changed during milking sessions. These findings indicated that the miRNA cargo within milk EVs remains stable irrespective of the time of day, suggesting their potential utility as diagnostic markers for mammary gland health.