In order to examine concentrations of cerebrospinal fluid (CSF) neurochemicals, the technique of lumbar puncture is typically used. However, the effect of the intrinsic stress of undergoing a lumbar puncture on CSF monoamine concentrations in humans has not yet been established. We used lumbar puncture followed 3 h later by continuous CSF sampling to examine the effect of lumbar puncture on levels of the dopamine and serotonin metabolites homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), respectively. Additionally, we examined the effect of lumbar puncture on the CSF HVA to 5-HIAA ratio. Immediately post lumbar puncture, CSF concentrations of HVA and 5-HIAA were, respectively, only 51 and 54% of the mean levels detected hours later. However, the HVA to 5-HIAA ratio remained stable during lumbar puncture. While HVA and 5-HIAA levels in CSF obtained via lumbar puncture reflect highly variable responses to the stress of the procedure, the ratio of these metabolites is unaffected.