Exosomes are nanometer sized vesicles (30-100 nm), released from cells following the fusion of multivesicular bodies with the plasma membrane. Exosomes have a major role in intracellular communication, immune response modulation, physiological and pathological functions, and an important use in different drug therapies. Moreover, they have been identified as potential biomarkers and are involved in cancer and many other disease processes. Despite the developments in this area, no studies exist that assess the variability of plasma exosomes measures within and between subjects. The purpose of this study was to assess within and between day variability of plasma exosomes concentration in healthy individuals. Also, the stability of exosomes during freeze/thawing cycles was measured in this study. Eleven healthy men were assessed for reliability of exosome concentrations taken over the same sampling period, over 10 hours of a day and over five days. Exosomes were isolated by differential ultracentrifugation and characterized by their size distribution and morphology by electron microscopy and using the Nanosight tracking analysis respectively. Mean plasma exosome values were1.505-2.245 × 10^8/mL). Within day variability was not significantly different (P = 0.95), and between day variability was not significantly different (P = 0.42). The interclass correlation coefficients (ICC) of 0.80 for within sample showed good reliability; ICC of 0.84 for between day plasma exosomes concentrations showed good reliability, and the within day ICC of 0.70 showed a moderate reliability. There was no significant difference observed in using fresh or frozen plasma in exosome quantification or exosome concentration. The statistics show that there is little significant variability in plasma exosome concentrations taken over a single sampling period, over a day or between days, which is vital for plasma exosome studies.

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