The objective of this prospective controlled study was to assess the effect of acupuncture (ACU) on acoustic myography (AM) of the middle gluteal muscle at the walk and trot in healthy sport horses (n=5). Acoustic myographic sensors were placed on the surface of the right and left middle gluteal muscles. Recordings were taken while the horses were walked and trotted in hand, in a straight line, before and immediately after ACU treatments, and 48 hours and one week afterwards. Acoustic myographic data was used to calculate an acoustic myographic (EST) score. Due to the small number of horses in this pilot study, data was presented descriptively. Gait abnormalities and sensitivity at routine ACU points were recorded at each session. All horses tolerated the procedure well and did not exhibit any signs of lameness at any time. Sensitivity at ACU points was present initially in 3 horses and was abolished immediately after ACU in all of them. All horses had an increased EST score after 48 hours, of which 3 had a persistent increase for 1 week afterwards, suggesting a greater speed of activation of muscle fibers after ACU. AM is a non-invasive technique, which is well tolerated and easily performed in a routine clinical setting. This small pilot study suggests a change in acoustic myographic data after ACU treatment but this will need to be confirmed in a greater number of horses.