There is an increasing demand for biomarkers in clinical treatment trials to demonstrate target engagement and to support disease modification claims. To be able to detect treatment related effects, a prerequisite is that the levels of the biomarker are stable over time or that the change over time is known. In the present study, the stability of α- and β-cleaved soluble amyloid-β protein precursor (sAβPPα and sAβPPβ), Aβ1-40 together with the phosphorylated form of neurofilament heavy/medium (pNfH/M) in cerebrospinal fluid (CSF) was analyzed in a cohort of 51 patients with Alzheimer's disease. In addition, the stability of Aβ1-40, Aβ1-42, and sAβPPβ in plasma was explored. Plasma and CSF was sampled at baseline and after 6-months follow up, and all patients were on stable treatment with acetylcholinesterase inhibitors. During this 6-month longitudinal follow-up, we saw a small, but consistent and statistically significant increase in CSF levels of sAβPPβ (103% of baseline levels) and a statistically significant decrease in the CSF levels of pNfH/M (91% of baseline levels). The mean level of the CSF biomarkers were very stable between baseline and endpoint, with within-patients coefficients of variation (CVs) of 5.84–17.3%, while the variability was larger for the plasma biomarkers, with CVs of 14.1–42.3%. This stability suggests that these biomarkers may have the potential to detect and monitor biochemical changes induced by disease-modifying drugs.