Blood donation is an essential step in transfusion medicine that must take into account the donor's welfare, collection effectiveness, and blood product quality. This prospective study enrolled 13 canine blood donors, each subjected to both gravity and suction collection methods, in a randomized order. Clinical parameters, including heart rate (HR), respiratory rate (RR), systolic blood pressure (SBP), and rectal temperature (RT), were evaluated at four time points, including when the donor was on the floor and on the collection table, and before and after blood donation. The number of times the donor and needle required repositioning, the duration of the donation, the noise created by the apparatus, and the presence of a hematoma were evaluated. The weight, index of hemolysis, and hematocrit of each unit of blood were recorded. There was no significant difference between collection methods for either the clinical parameters at each time point or the prevalence of hematoma formation, the frequency of needle repositioning, the hemolysis index, or hematocrit. Collection by suction was noisier (P < 0.0001), faster (P = 0.004), and associated with significantly less donor repositioning (P = 0.007). Suction appears to be a safe and cost-effective method that should be considered to optimize blood donation.