MicroRNAs are emerging as promising biomarkers for diagnosis of various diseases. Notably, cerebrospinal fluid (CSF) contains microRNAs that may serve as biomarkers for neurological diseases. However, there has been a lack of consistent findings among CSF microRNAs studies. Although such inconsistent results have been attributed to various technical issues, inherent biological variability has not been adequately considered as a confounding factor. To address this critical gap in our understanding of microRNA variability, we evaluated intra-individual variability of microRNAs by measuring their levels in the CSF from healthy individuals at two time points, 0 and 48 hours. Surprisingly, the levels of most microRNAs were stable between the two time points. This suggests that microRNAs in CSF may be a good resource for the identification of biomarkers. However, the levels of 12 microRNAs (miR-19a-3p, miR-19b-3p, miR-23a-3p, miR-25a-3p, miR-99a-5p, miR-101-3p, miR-125b-5p, miR-130a-3p, miR-194-5p, miR-195-5p, miR-223-3p, and miR-451a) were significantly altered during the 48 hours interval. Importantly, miRNAs with variable expression have been identified as biomarkers in previous studies. Our data strongly suggest that these microRNAs may not be reliable biomarkers given their intrinsic variability even within the same individual. Taken together, our results provide a critical baseline resource for future microRNA biomarker studies.