Cerebrospinal fluid (CSF) is a potential source of biomarkers for many disorders of the central nervous system, including Alzheimer disease (AD). Prior to comparing CSF samples between individuals to identify patterns of disease-associated proteins, it is important to examine variation within individuals over a short period of time so that one can better interpret potential changes in CSF between individuals as well as changes within a given individual over a longer time span. In this study, we analyzed 12 CSF samples, composed of pairs of samples from six individuals, obtained 2 weeks apart. Multiaffinity depletion, two-dimensional DIGE, and tandem mass spectrometry were used. A number of proteins whose abundance varied between the two time points was identified for each individual. Some of these proteins were commonly identified in multiple individuals. More importantly, despite the intraindividual variations, hierarchical clustering and multidimensional scaling analysis of the proteomic profiles revealed that two CSF samples from the same individual cluster the closest together and that the between-subject variability is much larger than the within-subject variability. Among the six subjects, comparison between the four cognitively normal and the two very mildly demented subjects also yielded some proteins that have been identified in previous AD biomarker studies. These results validate our method of identifying differences in proteomic profiles of CSF samples and have important implications for the design of CSF biomarker studies for AD and other central nervous system disorders. Cerebrospinal fluid (CSF) 1 is produced mainly by the choroid plexus within the ventricles of the brain. It circulates through the ventricular system and around the outside of the brain and spinal cord in the subarachnoid space. CSF is clinically accessible through standard lumbar puncture techniques. Because it is in direct contact with the brain interstitial fluid, biochemical changes taking place in the brain are often reflected in CSF. These features make CSF potentially a very useful source of biomarkers for diagnosis and response to treatment as well as for providing information on pathological processes underlying a number of central nervous system disorders, including Alzheimer disease (AD) (for reviews, see Refs. 1 and 2). Based on known pathology and the hypothesized etiology of AD, a few AD biomarkers in CSF have been identified that may differentiate groups of individuals with clinical disease from those who are cognitively normal, including amyloid  (A) 42, total tau, phospho-tau, isoprostane, and sulfatide (3-7). Although some candidate biomarkers have shown promise, each has its limitations. Researchers are still searching for biomarkers that have a higher sensitivity and specificity to differentiate AD from normal aging and other dementias, especially in the early stages. It would also be especially useful to develop antecedent biomarkers for AD, considering its estimated preclinical phase of 10 -20 years (8).Ne...