Objectives Cerebrospinal fluid (CSF) is a clear, colorless body fluid filling the central nervous system. The determination of the CSF total protein (TP) content represents an important screening test of various pathologies. We aimed to address the effect of sex and age on CSF TP content and the use of the current upper reference limits (URLs). Methods CSF TP content was analysed in a selected population of 1,252 patients (648 women and 604 men; age 18-89 years) who underwent lumbar puncture as a part of the diagnostic work-up. Samples presenting (i) more than 5 white blood cells (WBC)/µL, (ii) discolorations and (iii) reduced glucose were not included. Results The CSF TP content median values were significantly higher in men than in women (46 vs. 37 mg/dL) even after adjusting for age and different hospital inpatients. CSF TP content positively correlated with age both in men and in women with a constant difference between sexes of 8.5 mg/dL. Applying the most used URLs (mainly 45 and 50 mg/dL, but also 60 mg/dL), men received a laboratory report suggestive of altered CSF TP content more frequently than women. The use of age- and sex-calibrated CSF TP URLs reduced, but not eliminated, this sex-gap. Conclusions Using the current URLs, a condition of "elevated CSF TP content" may be overestimated in men or, conversely, underestimated in women, regardless of the age and of the diagnosis. These results highlighted the need to apply CSF TP URLs values normalized for both sex and age.