Selective M muscarinic acetylcholine receptor (mAChR) agonists are being developed as symptomatic treatment for neurodegenerative and neuropsychiatric disorders that lead to cognitive dysfunction. Demonstrating cognition enhancing effects in early phase clinical development in healthy subjects is difficult. A challenge with the M mAChR antagonist biperiden could be used to demonstrate procognitive and pharmacological effects of selective M mAChR agonists. The aim of this study was to develop such a model. To this end, twelve healthy elderly subjects participated in a randomized, placebo controlled, three-way crossover study investigating tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) effects of 2 and 4 mg biperiden. Repeated PD assessments were performed using neurocognitive tasks and electrophysiological measurements. A population PK-PD model was developed. Four mg biperiden showed significant impairment of sustained attention (-2.1% point in adaptive tracking (95% CI [-3.043; -1.148])), verbal memory (2-3 words fewer recalled (95% CI [-5.9; -0.2])), and working memory (up to 50 ms increase in the n-back task reaction time (95% CI [21.854; 77.882])) compared with placebo. The PK data was best fitted by a 2-compartment model and showed a high inter-occasion and inter-subject variability. Population PK-PD analysis quantified significant concentration-effect relationships for the n-back reaction times, n-back accuracies and adaptive tracking. In conclusion, biperiden caused M mAChR related dose- and concentration-dependent temporary declines in cognitive functioning. Therefore a biperiden pharmacological challenge model can be used for proof-of-pharmacology studies and to demonstrate cognition enhancing effects of new cholinergic compounds that are being developed. Clinical trial registration Netherlands Trial register identifier NL7146 This article is protected by copyright. All rights reserved.