OBJECTIVE: To determine whether cerebrospinal fluid (CSF) biomarkers for Alzheimer disease fluctuate significantly over time in a cohort of older, mildly symptomatic individuals. DESIGN: Biomarker validation in a clinical cohort. SETTING: University hospital inpatient unit. PARTICIPANTS: Ten patients admitted for CSF drainage for diagnostic purposes. MAIN OUTCOME MEASURES: The CSF levels of Aβ1-40, Aβ1-42, tau, and phosphorylated tau on threonine 181 (p-tau(181)) were measured every 6 hours for 24 or 36 hours. RESULTS: The mean coefficient of variation values for each biomarker assessed in our 10 patients were 5.5% (95% CI, 3.8%-10.0%) for Aβ1-42, 12.2% (9.0%-24.2%) for Aβ1-40, 8.2% (5.7%-15.1%) for total tau, and 11.9% (8.5%-23.0%) for p-tau(181). These values are only slightly higher than the variability in the assay. In addition, no significant circadian fluctuation in any Alzheimer disease biomarker was observed given the limitations of our sampling frequency. CONCLUSION: In a cohort of elderly patients, little fluctuation in the levels of important Alzheimer disease biomarkers in lumbar CSF is seen as a function of time.