Abstract: Objective To analyze the quantity and size distribution of 24-hour urinary extracellular vesicles (uEVs) from healthy
adults. Methods The 24-hour uEVs from 9 healthy adults were isolated by hydrostatic filtration dialysis (HFD). The
effectiveness of uEVs enrichment was evaluated using Western blotting and transmission electron microscopy (TEM). The
quantity and size distribution of the uEVs was analyzed with BCA protein quantification, TEM, and nanoparticle tracking
analysis (NTA). Results uEVs with different sizes and morphologies were observed under TEM. Western blotting confirmed
the expression of TSG101 in all the uEV fractions from the 9 donors, ranging from 132.50 to 760.70 ng/mL. NTA results showed
that the number of 24-hour uEVs amount ranged from 3.56 × 1012 particles to 5.12 × 1012 particles, with a CV of 14.23%. The
proportion of the vesicles with a diameter <40 nm was 0.04%-0.69% with a number range of (1.80-26.49) × 109 particles; the
proportion of vesicles with a diameter of 40-100 nm (which is consistent with the size of exosomes)was 22.07%-42.08% with a
number range of (1.00-1.77)×1012 particles. The proportion of vesicles with a diameter of 100-1000 nm (consistent with the size
of microvesicles) was 57.88%-77.85% with a number range of (2.09-3.86) × 1012 particles. Conclusion The established HFD
method allows efficient and convenient isolation of uEVs from a large amount of urine samples. The 24-hour uEVs from
healthy adults show narrow differences between individuals and thus can be an ideal source of samples for relevant studies.