Cerebrospinal fluid (CSF) is commonly used for assessing biomarkers of drug efficacy or disease progression in the central nervous system. Studies of CSF from pre-clinical species can characterize biomarkers for use in clinical trials. However, obtaining CSF from pre-clinical species, particularly rodents, can be challenging due to small body sizes, and consequently, low volumes of CSF. Surgical cannulation of rats is commonly used to allow for CSF withdrawal from the cisterna magna. However, cannulae do not remain patent over multiple days, making chronic studies on the same rats difficult. Moreover, CSF biomarkers may be affected by cannulation. Thus cannulation may contribute confounding factors to the understanding of CSF biomarkers. To determine the potential impact on biomarkers, CSF was analyzed from cannulated rats, surgically implanted with catheters as well as from non-cannulated rats. Brain protein biomarkers (αII-spectrin SBDP150 and total tau) and albumin, were measured in the CSF using ELISA assays. Overall, cannulated rat CSF had elevated levels of the biomarkers examined compared to non-cannulated rat CSF. Additionally, the variation in biomarker levels observed among CSF from cannulated rats was greater than that observed for non-cannulated rat CSF. These results demonstrate that in some cases, biomarker assessment using CSF from cannulated rats may differ from that of non-cannulated animals and may contribute confounding factors to biomarker measurements and assay development for clinical use.