This study compared the effects of exercise or TXB2 and beta-TG when evaluated by four methods: 1) not adjusted; 2) adjusted for plasma volume changes (PV); 3) standardized per 10(5) platelets (PC); 4) or both PC and PV (PC-PV). Blood was collected from 16 men (41.3 +/- 8.1 yr) at rest after 30 min of exercise (IPE) and after 30 min recovery. Resting TXB2 and beta-TG concentrations were 62.0 +/- 6.2 pg.mL-1 and 129.8 +/- 12.5 ng.mL-1, respectively. When expressed on a per 10(5) platelet basis, resting PCTXB2 was 23.8 +/- 2.8 pg.mL-1.10(5-1) platelets and PC beta-TG was 50.77 +/- 6.0 ng.mL-1. 10(5-1) platelets. At IPE, TXB2 decreased 20.5% and beta-TG increased 13.6%. Thirty minutes after exercise TXB2 was 4.2% lower than resting values, whereas beta-TG was 26% higher. TXB2, beta-TG, PVTXB2, and PV beta-TG were not significantly altered by exercise. The only significant changes in TXB2 occurred at IPE when values were adjusted for changes in platelet count. At IPE, PCTXB2, and PC-PVTXB2 decreased 32.8% and 33.6%, respectively (P < 0.05). Similarly, beta-TG were not altered significantly by exercise except when the samples taken after 30 min of recovery were adjusted for changes in platelet count. At 30 min post-exercise PC beta-TG and PC-PV beta-TG were 21.2% and 28.4% greater (P < 0.05) than the resting beta-TG values. These data suggest that methods used to adjust concentrations of platelet derived substances for changes owing to exercise may influence conclusions about the effect of exercise on platelet function. Thus, it is imperative that researchers consider the purpose for which they are collecting TXB2 and beta-TG, as well as other constituents derived from blood cells, before they determine which methods of analysis to use.