Background This study was aimed to define the short- and medium-term biological variation (BV) estimates, the index of individuality and the reference change value (RCV) of platelet count, platelet distribution width, mean platelet volume, platelet larger cell ratio, plateletcrit and immature platelet fraction. Methods The study population consisted of 43 health subjects, who participated to the assessment of medium-term (21 subjects; blood sampling once a week for 5 consecutive weeks) and short-term (22 subjects; blood sampling once a day for 5 consecutive days) BV study, using Sysmex XN-module. Eight subjects were also scheduled to participate to both phases. The data were subject to outlier analysis prior to CV-ANOVA, to determine the BV estimates with the relative confidence intervals. Results The medium-term and short-term within-subject BV (CV I ) was comprised between 2.3 and 7.0% and 1.1–8.6%, whereas the medium-term and short-term between-subjects BV (CV G ) was comprised between 7.1 and 20.7% and 6.8–48.6%. The index of individuality and index of heterogeneity were always r espectively < 0.6 and > 0.63 for all the parameters, in both arms of the study. The RCVs were similar for all parameters, in both arms of the study. Conclusion This study allowed to define the BV estimates of many platelet parameters, some of them unavailable in literature. The kinetics of platelet turnover suggests the use of short-term BV data for calculating analytical goals and RCV. The correct clinical interpretation of platelet parameters also necessitates that each laboratory estimates local RCV values. © 2017 Elsevier B.V.