Simple SummaryNutrition is one of the main causes of thyroid response and energetic metabolism. Presently, there is a lack of information on the physiological effect of moderate activities in canines, particularly in guide dogs. Aim was to compare the effect of diet on thyroid and lipid status in guide dogs, during a 12-weeks training, fed two commercial diets, one, HPF, characterized by low-carbohydrate/high-protein/high-fat (29:39:19% as fed) and the other, LPF, characterized by high-carbohydrate/low-protein/low-fat (50:24:12% as fed) content. Our hypothesis was that the intake of a diet rich in fat and protein would have given a better response than the carbohydrate-rich diet for thyroid and lipid homeostasis to cope with the increased energy demands of dogs subjected to the training period. Results evidenced that the consumption of a diet rich in fat and protein appears the nutritional plan most suitable to support moderate exercise for guide dogs during the training work; this diet supports maintenance of body weight, Body Condition Score (BCS), and serum baseline thyroid and lipid profiles, offering potential improvements in dogs’ performances. However, the chronic ingestion of diets that are extreme in their composition of either fat or carbohydrate should be always approached with caution.AbstractNutrition plays a leading role that most influences thyroid response and energetic metabolism. Aim was to compare the effect of diet on thyroid and lipid status in guide dogs during a 12-weeks training period. Eight Labrador Retrievers were divided into two groups homogeneous for sex, age, body weight, and Body Condition Score (BCS) and fed two commercial diets one, HPF, characterized by low-carbohydrate/high-protein/high-fat (29%:39%:19% as-fed) and the other, LPF, by high-carbohydrate/low-protein/low-fat (50%:24%:12% as-fed) content. The serum thriiodothyronine (T3), thyroxine (T4), cholesterol (CHOL), triglycerides (TAGs) and non-esterified fatty acids (NEFA) were determined at Day 0, 28, 56, and 84, before the daily training. Statistical model included the effects of Diet (HPF vs. LPF) and Time (Day 0 to Day 84), and their interaction. In the HPF group, Diet significantly (p < 0.01) increased T4, CHOL, and TAGs and decreased NEFA. In both groups, Time significantly (p < 0.05) increased T4 and TAGs, CHOL at Day 28, and NEFA at Day 56. The interaction did not influence serum hormones and lipid pattern. The adjustments in thyroid and lipid responses to moderate exercise in HPF group were driven mainly by the nutrient composition of the diet in relation to the involvement of metabolic homeostasis.