Lumbar puncture (LP) is commonly used in the diagnostic workup of neurological patients, often to exclude inflammatory diseases of the central nervous system. In clinical practice, an increase of white blood cell count (WBC) in the cerebrospinal fluid (CSF) after a LP is often assumed as reactive to the first puncture. Scientific evidence of this hypothesis, however, is lacking. Retrospective review of laboratory parameters was done by analyzing CSF of patients who had at least two LPs between 2012 and 2016 in a single center. Inclusion criteria were a normal CSF WBC in the first LP as well as absence of any underlying disease typically associated with increased CSF WBC. A total of 176 patients (age 57.0 ± 17.6) with 260 serial LPs were included. No significant effect on the CSF WBC (1.2 ± 1.1 vs 1.4 ± 1.4/μl, p = .17), lactat and protein level between consecutive punctures was found after a second LP. In the subgroup of 104 patients who had two LPs within ten days, only one (0.96%) showed a mild abnormal CSF WBC (9 leukocytes/μl) in the second LP. A raise of CSF WBC after LP is rare and not commonly found; therefore, it should lead to careful exclusion of other, especially inflammatory diseases. The needle size is important to minimize the trauma during LP and seems to have an influence on the rate of reactive increase of CSF WBC after LP.