The purpose of this study was to evaluate the effects of botulinum toxin A (BTX-A, Botox) dilution volume and post-injection exercise with electrical stimulation on muscle paralysis. We injected 10 units of BTX-A diluted with 0.1 ml (B1, n=8) or 0.5 ml (B5, n=8) normal saline into both gastrocnemius muscles of 16 New Zealand white rabbits; two controls received no BTX-A. After BTX-A injection, all rabbits received calf muscle stretching exercise and electrical stimulation for 2 hours on the left leg. The compound muscle action potential (CMAP) decrease was most pronounced at 1 week and progressive recovery was observed (i.e. recovery from paralysis, increase of CMAP). There was a significant decrease of CMAP amplitudes in the B5 group compared with the B1 group at week 1 and week 4 (p<0.001). Left limbs with stretching exercise and electrical stimulation showed lower CMAP amplitudes compared with control right limbs of all rabbits. To maximize the muscle paralysis effect of BTX-A, increasing dilution volume and performing post-injection stretching exercise with electrical stimulation may be a promising strategy for increasing the beneficial effect of BTX-A treatment. Future studies are needed to investigate the clinical application of this finding.