Prolonged multiday exercise in dogs is often associated with a caloric deficit that can lead to changes in body composition but may also result in an increase in lean body mass due to a conditioning effect. To test the hypothesis that multiday exercise decreases fat mass but increases lean body mass, body composition was measured using deuterium oxide dilution in a team of racing Alaskan sled dogs competing in a multiday race. Twelve dogs completed 961 km in 6 days, resulting in a 3% decrease in body mass (P=0.003) and 16% decrease in body fat (P=0.005), and an increase in total body water as a fraction of body mass (P=0.01). Within the study population, there was considerable dog-to-dog variation, with 2 dogs gaining fat mass and 2 dogs losing over 40% of their pre-race fat mass during the measurement period. These results confirm that dogs participating in multiday exercise events are at risk for loss of body fat reserve, likely due to a net calorie deficit. However, individual dog responses to an exercise challenge can be highly variable, emphasizing the need for careful assessment of individual dogs.