Background <p>Platelet-rich plasma (PRP) is extensively used in regenerative medicine; however, its preparation lacks standardization. The difference in rotor angle between fixed-angle and horizontal rotors influences PRP quality. This study aims to compare the effects of these two rotor types on PRP preparation.</p> Methods <p>A total of 15 subjects were enrolled in the study, with each subject donating 80 ml of whole blood, which was then divided into two separate tubes, each containing 40 ml. PRP was prepared using two rounds of centrifugation with both fixed-angle and horizontal rotors under optimal parameters. Platelet recovery, concentration, growth factor content, and effects on cell co-culture were assessed.</p> Results <p>The fixed-angle centrifuge demonstrated higher platelet recovery efficiency, increased platelet content, and greater PDGF-BB levels in the PRP, with no significant change in CD62P expression. Co-culture assays showed enhanced cell proliferation and migration with PRP prepared using the fixed-angle rotor compared to the horizontal rotor.</p> Conclusions <p>Under appropriate centrifugation parameters, the fixed-angle centrifuge can extract higher quality PRP in a shorter time.</p> Level of Evidence II <p>This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors <a href="http://www.springer.com/00266">www.springer.com/00266</a>.</p>