Background: Cerebrospinal fluid (CSF) measures of β-amyloid1–42 and tau differ between patients with Alzheimer’s Disease (AD) and elderly normal controls. The effect of time and APOE genotype on these biomarkers continues to be elucidated. Methods: We assessed CSF β-amyloid1–42 and tau in 20 mild-to-moderate AD patients, 11 APOE Ε4+ and 9 APOE Ε4–, over a mean time of 3.8 years (range 1–11.1 years). Results: Over the period measured, CSF β-amyloid1–42 levels were lower in APOE Ε4+ compared to APOE Ε4– patients, and the levels decreased over time. Tau levels were stable over time and did not show an effect of APOE allele. Conclusions: While this is a limited clinical sample, the further decrease in CSF β-amyloid1–42 (i.e., more abnormal) combined with the CSF tau stability over a mean period of almost 4 years suggests that β-amyloid1–42 and tau maintain their potential usefulness as diagnostic biomarkers over time. These findings should be taken into account if CSF β-amyloid1–42 and tau are used as measures of treatment response.