Abstract Objectives The aim of this study was to evaluate the impact of long-term sample storage on the concentrations of neopterin and neurofilament light (Nfl) in cerebrospinal fluid (CSF) samples. These are useful markers of neuroinflammation and neuronal damage and have been applied as biomarkers for several neurological diseases. However, different pre-analytical variables have potential to influence results. Methods Twenty-one CSF samples donated by patients with HTLV-1-associated myelopathy (HAM) and stored for up to 11 years at −80 °C were retested after three-years for neopterin (n=10) and Nfl (n=11) by ELISA. Results There was a strong correlation between the paired results (r>0.98, p<0.0001). Neopterin concentrations (nmol/L) ranged from 12.4 to 64 initially and from 11.5 to 64.4 when retested, with means (SD) of 30 (18.4) 1st test and 33 (19.1) 2nd test. Nfl concentrations (pg/mL) ranged from 79.9 to 3,733 initially and from 86.3 to 3,332, when retested with means (SD) of 1,138 (1,272) 1st test and 1,009 (1,114) at re-test. Conclusions Storing CSF samples at −80 °C appears not to impact the quantification of neopterin and Nfl allowing confidence in the reporting of archived samples.