Horses competing in cross-country tests are subjected to high physical demands. Within the scope of this prospective longitudinal study, blood values of 20 elite eventing horses were examined before and after two- to four-star cross-country rides. The aim was to find out whether blood-based markers for skeletal muscle and cardiac muscle function change after cross-country exercise. Parameters that provide information about fluid balance, muscle enzymes, metabolites and cardiac muscle-specific markers were investigated. We developed an approach to eliminate the concentration changes caused by reduced plasma volume. Parameters were measured pre, 10 and 30 min post exercise and the next morning and were evaluated using a mixed model. Thirty minutes after exercise, most parameter concentrations changed in an exercise-dependent manner. The next morning, most exercise-related markers recovered rapidly, while creatine kinase (CK) (26% increase; p = 0.008) and lactate dehydrogenase (LDH) (15% increase; p < 0.001) showed a declining but sustained increase. Cardiac troponin I (cTnI) increased above the reference range in 40 of the 55 rides (73%) and in 18 of 20 horses in the morning after exercise.