Mean platelet volume (MPV), a measure of platelet size, has previously been noted to change in a time dependent manner in ethylene-diaminetetraacetic acid (EDTA) and citrate anticoagulants. We measured MPV in healthy volunteers using several different anticoagulants and at different temperatures. In ten volunteers MPV was measured (Sysmex NE 8000 autoanalyser) serially over a 29 h period in 15 different anticoagulant/antiplatelet combinations kept at room temperature and on ice-water mixture. We evaluated the following mixtures: K3EDTA, K3EDTA/prostaglandin E1 (PGE1), K3EDTA/PGE1/theophylline, Na2EDTA, Na2EDTA/PGE1, citrate 1:9, citrate 1:4, citrate 1:9/PGE1/theophylline, citrate 1:4/PGE1/theophylline, citrate 1:9/PGE1, citrate 1:9/dextrose, citrate 1:4/dextrose, acid-citrate-dextrose (ACD) 15%, ACD 11.9%, ACD 11.9%/Na2EDTA. In a second experiment we evaluated MPV, at room temperature only, in a further 20 volunteers in K3EDTA, citrate 1:9, ACD 11.9%/Na2EDTA, ACD 11.9% (total = 30) and citrate-phosphate-dextrose-adenine (CPDA; total = 20). Over the 29 h period there was a significant positive trend of increasing MPV in K3EDTA, CPDA and ACD 11.9%/Na2EDTA and a negative trend in citrate 1:9. ACD 11.9% showed no significant change in MPV with time at room temperature. This methodology should enable the wider application of MPV measurement for routine clinical and research studies.