Recently, we proposed that the higher stroke volume (SV) and cardiac output (Q) of endurance-trained (ETR) versus untrained (UTR) individuals are attributable primarily to the enhanced diastolic filling of ETR consequent to a larger blood volume (BV). To test this hypothesis, we examined the effects of manipulating BV on the cardiac function of six ETR and six UTR males. Both groups were examined in the control BV condition (BVctl), then ETR were examined immediately following a 500 mL reduction in BV (BVred) and UTR were examined immediately following a 500 mL expansion of BV (BVexp). In BVctl, compared with UTR, ETR had significantly greater BV (16%), maximal diastolic filling rate (47.4%), maximal ventricular emptying rate (24.6%), SVmax (31.6%), Qmax (29%) and VO2max (54.5%). Following BVexp in UTR, there were immediate significant increases in maximal diastolic filling rate (22.5%), SVmax (9.1%), Qmax (8.9%), and VO2max (12.7%). Following BVred in ETR there were immediate significant decreases in maximal diastolic filling rate (27%), SVmax (14.3%), Qmax (14.7%), and VO2max (7.0%). Maximal systolic emptying rate did not change significantly following BVred or BVexp. We conclude that changes in SV and Q consequent to alterations in BV are attributable primarily to changes in diastolic function, and the majority of the higher diastolic filling rate of ETR is due to their larger BV.