The purpose of this study was to 1) evaluate and compare the athletic performance of 14 horses and 16 mules, working at high altitude (2700 to 4000 m) in the course of a commercial pack station routine and 2) assess the effect of fat supplementation (10% by weight) on performance. Lactate, haemoglobin, glucose and free fatty acid (FFA) concentrations were measured on Day 1 (prior to supplementation, animals at rest) and then each day thereafter from day 3–7 (i.e. a total of 6 samples for each animal) after an exercise test. Exercise tests ranged in power requirements from 160–730 J/s. Lactate concentrations were increased after the exercise bouts (P=0.029) and were positively correlated (r=0.93) to power. However the value for lactate concentrations indicated that these animals were working well below maximal intensities (mean ± s.e. 1.19 ± 0.1 mmol/l at the most intense work load) and there were no differences between mules and horses. Haemoglobin concentrations were also correlated with power (r=0.79). Glucose and FFA concentrations were increased after exercise. Fat supplemented animals maintained blood glucose concentrations higher than controls and had higher FFA concentrations after the first exercise bout. The only difference between mules and horses was noted after the most intense exercise period: FFA concentrations were higher in mules than horses during exercise.