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Spinal serotonin enables neuro‐motor recovery (i.e., plasticity) in patients with debilitating paralysis. While there exists time of day fluctuations in serotonin‐dependent spinal plasticity, it is unknown, in humans, whether this is due to dynamic changes in spinal serotonin levels or downstream signaling processes. The primary objective of this study was to determine if time of day variations in spinal serotonin levels exists in humans. To assess this, intrathecal drains were placed in seven adults with cerebrospinal fluid (CSF) collected at diurnal (05:00 to 07:00) and nocturnal (17:00 to 19:00) intervals. High performance liquid chromatography with mass spectrometry was used to quantify CSF serotonin levels with comparisons being made using univariate analysis. From the 7 adult patients, 21 distinct CSF samples were collected: 9 during the diurnal interval and 12 during nocturnal. Diurnal CSF samples demonstrated an average serotonin level of 216.6 ±$ \pm $ 67.7 nM. Nocturnal CSF samples demonstrated an average serotonin level of 206.7 ±$ \pm $ 75.8 nM. There was no significant difference between diurnal and nocturnal CSF serotonin levels (p =.762). Within this small cohort of spine healthy adults, there were no differences in diurnal versus nocturnal spinal serotonin levels. These observations exclude spinal serotonin levels as the etiology for time of day fluctuations in serotonin‐dependent spinal plasticity expression. [ABSTRACT FROM AUTHOR]