On dairy farms, flooring is often sloped to facilitate drainage. Sloped floors have been identified as a possible risk factor for lameness, but relatively little is known about how this flooring feature affects dairy cattle. Ours is the first study to evaluate the short-term effects of floor slope on skeletal muscle activity, restless behavior (measured by number of steps), and latency to lie down after 90min of standing. Sixteen Holstein cows were exposed to floors with a 0, 3, 6, or 9% slope in a crossover design, with a minimum of 45h between each testing session. Electromyograms were used to evaluate the activity of middle gluteal and biceps femoris muscles. Muscle activity was evaluated in 2 contexts: (1) static muscle contractions when cows continuously transferred weight to each hind leg, before and after 90min of standing; and (2) dynamic contractions that occurred during 90min of treatment exposure. Median power frequency and median amplitude of both static and dynamic muscle electrical signals were calculated. Total muscle activity was calculated using the root mean square of the signals. Restless behavior, the number of steps per treatment, steps and kicks in the milking parlor, and the latency to lie down after the test sessions were also measured. It was predicted that restless behavior, muscle fatigue (as measured by median power frequency and median amplitude), total muscle activity, and latency to lie down after testing would increase with floor slope. However, no treatment differences were found. Median power frequency was significantly greater for the middle gluteal muscle [35±4Hz (mean and SE)] compared with the biceps femoris muscle (24±3Hz), indicating that the contractive properties of these muscles differ. The number of steps per minute and total muscle activity increased significantly over 90min of standing, irrespective of floor slope. Although restless behavior and muscle function did not change with slope in our study, this work demonstrates that electromyograms can be used to measure skeletal leg muscle activity in cattle. This technology, along with restless behavior, could be useful in assessing cow comfort in other situations, such as prolonged standing.