While the pathogenesis of Botulinum toxin type A (BTX-A)-induced muscle weakness has been systematically researched, little is known about the effects of motor fibre paralysis on the mechanical properties of skeletal muscle. Here, the long-term effect of BTX-A injection on the force-length and force-frequency properties of rabbit knee extensors is investigated. BTX-A-induced muscle weakness was greater at short compared to long muscle lengths and at low compared to high stimulation frequencies four weeks following intervention. Therefore, we conclude that the paralysing effects of BTX-A are not uniform, and must be considered in biomechanical models of musculoskeletal rehabilitation in which BTX-A is used therapeutically, as for example in patients with cerebral palsy. Although the present results could be explained through a variety of mechanisms, this study does not allow for drawing firm conclusions about the length and frequency-dependent effects of BTX-A.