Background: Reported concentrations of amyloid β (1-42) (Aβ42) and tau in cerebrospinal fluid (CSF) differ among reports. We investigated the effects of storage temperature, repeated freeze/thaw cycles, and centrifugation on the concentrations of Aβ42 and tau in CSF. Methods: Stability of samples stored at -80 °C was determined by use of an accelerated stability testing protocol according to the Arrhenius equation. Aβ42 and tau concentrations were measured in CSF samples stored at 4, 18, 37, and -80 °C. Relative CSF concentrations (%) of the biomarkers after one freeze/thaw cycle were compared with those after two, three, four, five, and six freeze/thaw cycles. In addition, relative Aβ42 and tau concentrations in samples not centrifuged were compared with samples centrifuged after 1, 4, 48, and 72 h. Results: Aβ42 and tau concentrations were stable in CSF when stored for a long period at -80 °C. CSF Aβ42 decreased by 20% during the first 2 days at 4, 18, and 37 °C compared with -80 °C. CSF tau decreased after storage for 12 days at 37 °C. After three freeze/thaw cycles, CSF Aβ42 decreased 20%. CSF tau was stable during six freeze/thaw cycles. Centrifugation did not influence the biomarker concentrations. Conclusions: Repeated freeze/thaw cycles and storage at 4, 18, and 37 °C influence the quantitative result of the Aβ42 test. Preferably, samples should be stored at -80 °C immediately after collection.