Abstract Objective—To use electromyography (EMG) to measure physiologic activity of the longissimus dorsi muscles of horses during trotting on a treadmill. Animals—15 adult horses (5 to 20 years old that weighed 450 to 700 kg) that did not have clinical signs of back pain. Procedure—Data were recorded for each horse during trotting on a treadmill at speeds of 2.6 to 4.4 m/s. Surface electromyography was recorded bilaterally from the longissimus dorsi muscles at the levels of T12, T16, and L3. Results—In each motion cycle, 2 EMG maxima were found at the end of the diagonal stance phases. The EMG activity peaked slightly later at L3 than at T12 and T16. Maximum EMG amplitudes were highest at T12 and decreased caudally, with mean ± SD values of 4.51 ± 1.20 mV at T12, 3.00 ± 0.83 mV at T16, and 1.78 ± 0.67 mV at L3. Mean minimum EMG activity was 1.30 ± 0.63 mV at T12, 0.83 ± 0.35 mV at T16, and 0.80 ± 0.39 mV at L3. The relative amplitudes (ie, [maximum – minimum]/maximum) were 67 ± 11% at T12, 66 ± 8% at T16, and 71 ± 8% at L3. Conclusions and Clinical Relevance—Activity of the longissimus dorsi muscles is mainly responsible for stabilization of the vertebral column against dynamic forces. The difference between minimum and maximum activity may allow application of this method as a clinical tool. Data reported here can serve as reference values for comparison with values from clinically affected horses. (Am J Vet Res 2004;65:155–158)