Objective: To determine the influence of transportation by road and air on heart rate (HR) and HR variability (HRV) in horses. Animals: 6 healthy horses. Procedures: ECG recordings were obtained from horses before (quarantine with stall rest [Q]; 24 hours) and during a journey that included transportation by road (RT; 4.5 hours), waiting on the ground in an air stall (W; 5.5 hours), and transportation by air (AT; 11 hours); HR was determined, and HRV indices of autonomic nervous activity (low-frequency [LF; 0.01 to 0.07 Hz] and high-frequency [HF; 0.07 to 0.6 Hz] power) were calculated. Results: Mean ± SD HRs during Q, RT, W, and AT were 38.9 ± 1.5 beats/min, 41.7 ± 5.6 beats/min, 41.5 ± 4.3 beats/min, and 48.8 ± 5.6 beats/min, respectively; HR during AT was significantly higher than HR during Q. The LF power was significantly higher during Q (3,454 ± 1,087 milliseconds2) and AT (3,101 ± 567 milliseconds2) than it was during RT (1,824 ± 432 milliseconds2) and W (2,072 ± 616 milliseconds2). During Q, RT, W, and AT, neither HF powers (range, 509 to 927 milliseconds2) nor LF:HF ratios (range, 4.1 to 6.2) differed significantly. The HR during RT was highly correlated with LF power (R2 = 0.979), and HR during AT was moderately correlated with the LF:HF ratio (R2 = 0.477). Conclusions and Clinical Relevance: In horses, HR and HRV indices during RT and AT differed, suggesting that exposure to different stressors results in different autonomic nervous influences on HR.