Seven untrained Standardbred horses were used in a training programme of 6 weeks to evaluate the effects of exercise and training on bone metabolism. The horses were exercised on a treadmill according to a standardized exercise test (SET 1: six incremental steps, 5 min duration each; start 5 m/s, increase 1 m/s). SET 1 was followed by a training programme of 6 weeks. In alternating order: high-speed exercise (HSE): 15 min duration, start at VLa4, continuous increase in speed every 60 s by 0.3 m/s (14 incremental steps); low-speed exercise (LSE): constant velocity at VLa2.5, duration: approximately 60-90 min (total training programme: eight HSE and eight LSE sessions). SET 2 finished the training programme and a deconditioning period of 12 weeks followed. Blood samples for lactate, total plasma protein (TPP), osteocalcin, and ICTP (cross-linked C-telopeptide of type I collagen) were collected. ICTP increased during SET 1 and SET 2, whereas osteocalcin decreased to below resting concentration 24 h after SET 1. A rise in ICTP was observed during LSE 1 and LSE 8, which was followed by a drop 24 h after exercise. No changes in osteocalcin were noted during LSE 1, but 24 h after LSE 1 osteocalcin dropped to below pre-exercise levels. LSE 8 resulted in an increase in osteocalcin, followed by a drop 24 h after LSE 8. Osteocalcin and ICTP were not affected by HSE. Baseline osteocalcin levels dropped during the course of training. The acute response of biochemical bone markers indicates a direct influence of a single bout of exercise on bone metabolism.