Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements.