The aims of the study were to clarify the impact of storage time and centrifugation strategy on brain-derived neurotrophic factor (BDNF) levels in human serum and plasma samples. In addition, we analyzed associations between BDNF levels, cardiorespiratory fitness and waist circumference. Seventeen healthy males (25.2 (4.1) years) were included in the study. Blood samples were drawn after an overnight fast and treated to different protocols, varying in time before centrifugation and centrifugation strategy. BDNF was analyzed in serum, normal plasma (NP) and platelet-poor plasma (PPP). Also, waist circumference and cardiorespiratory fitness were measured. A large increase was observed in serum BDNF levels during the first hour of clotting. BDNF in NP correlated with PPP, whereas no correlations were found between BDNF in serum and plasma. Though not statistical significant, correlations between fitness and BDNF in serum changed from positive at 30 min. to negative when clotting time was ≥60 min. In conclusion, BDNF levels in serum were affected by clotting time, whereas BDNF levels in plasma were influenced by centrifugation strategy. Importantly, BDNF in serum and plasma appears to reflect two different pools of BDNF. The biological relevance of the velocity of BDNF release during clotting and its dependence upon fitness must be investigated further.