Agility is physically demanding and dogs encounter a considerable risk of injury during training and competition. Pre-performance warm-up is used routinely among human athletes to prepare the tissues for these physical demands, but in canine sports evidence for effects of warm-up is lacking. The aim of this study was to investigate the effects of warm-up in dogs on two major muscles involved in locomotion. It was hypothesised that, after warm-up, the muscles would be used more efficiently (more fibre resting time/total time), recruit fewer fibres (reduced spatial summation) and/or activated with a lower firing frequency (reduced temporal summation). The following factors ‘sex, age, weight, height, training level and agility experience’ were evaluated for their potential impact on muscle function parameters. Fourteen large (≥46 cm at the withers) agility dogs of different breeds and training levels performed a 5 min warm-up program three times, with a 2 min break between the programs for recording purposes. Acoustic myography sensors were attached on the skin over the muscles m. triceps brachii (TB) and m. gluteus superficialis (GS). Recordings of muscle activity were made, while the dogs trotted before warm-up and after each 5 min warm-up program. The dogs used TB more efficiently after 5 min (p<0.05), 10 min (p<0.05) and 15 min (p<0.001) of exercise compared to pre-warm-up values. No changes were found in the activity of GS. For well-trained dogs, TB recruited fewer muscle fibres after 10 and 15 min of warm-up compared to dogs that trained less than 1 h weekly (p<0.03). For dogs with more than 2 years of experience, GS had a lower firing frequency before and after 10 min warm-up compared to dogs with less experience. The results indicate that warm-up alters muscle activation by an increased muscular efficiency. Training level and experience have an influence on muscle function parameters.