Assays in which samples of biological fluids are dispensed onto reagent-containing porous substrates are improved in the accuracy and repeatability by dispensing the biological fluids in two or more fractions thereof, separated by intervals in which the biological fluid is not dispensed. Reagents and other fluids may be dispensed during the intervals when the biological fluid is not dispensed. Alternatively, reagents and other fluids may be dispensed in a similar manner onto substrates already containing biological fluids.