Platelet‐rich fibrin membranes are biomaterials widely used for therapeutic purposes, and canonically produced through the processing of peripheral blood with fixed‐angle rotor centrifuges. In this work, we evaluate the in vitro stability and release of cytokines and growth factors when these biomaterials are produced with a horizontal swing‐out clinical centrifuge. Membranes produced from the blood of 14 donors were morphologically evaluated by scanning electron microscopy and fluorescence microscopy, and their stability was assessed by photographic recording after incubation in culture medium for up to 28 days. The release of 27 cytokines and growth factors was monitored for three weeks through a multiparametric immunoassay. The fibrin membranes presented complex three‐dimensional structure with a high density of nucleated cells. A large release of growth factors [platelet derived growth factor, fibroblastic growth factor (bFGF), and vascular endothelial growth factor] was detected in the first 24 h, followed by time‐dependent decay, maintaining significant concentrations after three weeks. Both anti‐inflammatory and pro‐inflammatory cytokines presented different release peaks, maintaining high rates of elution for up to 21 days. Chemokines of relevance in tissue repair [RANTES, granulocyte colony‐stimulating factor (G‐CSF)] were also produced in large quantities throughout the experimental period. The present results demonstrate that blood‐derived fibrin membranes with high structural stability and cell content can be generated by horizontal centrifugation, being able of a prolonged production/release of growth factors and pro‐ and anti‐inflammatory cytokines. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1373–1380, 2018.