High‐starch diets may affect equine hindgut microbiota and increase blood glucose levels, which may cause unwanted physiological changes, but may also elicit behavioural changes such as increased fear reactions. The purpose of the current study was to feed a high starch (300) and low fat (43; HS_LF) or a low starch (60) and high fat (85; LS_HF, g/kg of DM) concentrate within the available commercial range and investigate how muscle endurance and fear reactions of horses respond to different diets. Twenty Danish Warmblood stallions (4 years) were randomly allocated to two treatments: LS_HF (n = 10) and HS_LF (n = 10) for 9 weeks. During the two last weeks, a single step exercise test was performed, and plasma metabolites and blood gases were measured before and after exercise in a 2 × 2 factorial design. The effect of two diets on fearfulness was tested by exposing the horses to novel objects test (T1 and T2). Plasma metabolites was not affected by diets. However, plasma level of glucose post‐exercise (4.9) was lower than pre‐exercise (5.6 mmol/L; p < 0.001). Similarly, plasma level of insulin post‐exercise (4.2) was lower than pre‐exercise (13.1 pmol/L; p < 0.001). Plasma level of lactate dehydrogenase (p < 0.001), non‐esterified fatty acids (p = 0.002), β‐hydroxybutyrate (p = 0.001), and fructosamine (p = 0.01) post‐exercise was higher than pre‐exercise. Regardless of type of diets, RRR‐α‐tocopherol was the dominance α‐tocopherol stereoisomers in plasma. In conclusion, during aerobic exercise, fat to starch ratio in horse diets within the normal range had no significant effect on plasma metabolites. However, horses fed LS_HF tended to show more investigative behaviour than horses fed HS_LF. [ABSTRACT FROM AUTHOR]