Abstract In humans, meal size has been shown to affect heart function with meal ingestion being associated with an increase in heart rate (HR) and cardiac output lasting significantly longer when larger meals are consumed. In horses, HR spikes at the sight of feed due to anticipation, but no studies have specifically evaluated the effect of meal frequency or size on HR. Heart rate has also been used as a measure both positive and negative stress. Domesticated horses are often meal fed, with small frequent meals requiring more labor than large infrequent meals. Fewer larger meals may have negative welfare implications for a grazing species. As part of a larger project investigating the effects of meal frequency on the behavior and metabolism of horses, the objective of this study was to evaluate equine HR in response to feeding frequencies commonly practiced in the field [once (1x), twice (2x), or thrice (3x) per day]. Feeding times were at 0800 (1x, 2x, and 3x), 1300 (3x), and 1800 (2x and 3x) h. The 12 adult clinically healthy horses [average ± SD; 14 ± 6 yr and 572 ± 32 kg body weight (BW)] were separated into two equal groups before completing a 14-d acclimation period to the diet during which they were fed 2x. The diet was provided at 2% BW on a dry matter (DM) basis (1.55% BW grass hay in a slow feed hay net plus 0.45% BW complementary fortified feed). During the study the same diet was fed but the meal frequency, and therefore meal size, differed between the treatments. The order of treatments, lasting 1 wk each, was decided at random for each group. On d 6 of each treatment HR was measured using individual H10 Polar HR monitors, which recorded every second from 0700 to 1900 h yielding 1,514,799 data points. During this time the aisleways housing the stalled horses were blocked off to minimize exposure to human presence other than at feeding time(s). After removing outliers, the mode was calculated for HR of each horse, which was considered to be the resting HR for that horse and ranged from 31 to 46 beats/min. The percentage of data above, below, or at the mode was calculated for each horse over the 12 h period and evaluated using Chi Square tests for statistical significance. The proportion of HR values above, below, or at the mode differed between treatments (P = 0.02). When fed 1x, 69 ± 14% of the HR readings were above the mode on average, as compared with 42 ± 18% and 44 ± 18% for 2x and 3x, respectively. These results suggest that the increase in HR was more than anticipatory and that there may be sustained stress when horses are fed at fewer frequency, such as once daily.