Ageing is associated with a gradual bone loss and physical activity has been suggested as practical strategy for a non-pharmacological prevention of osteoporosis. However, until now, the specific mechanism by which physical activity affects bone tissue is not thoroughly understood. The aim of this study was to evaluate the effect of strenuous exercise on bone metabolism as a function of age and fitness level. Eighteen physically highly active elderly participants (mean age 71.7+/-7.3 years, HAcEl group), 18 moderately active elderly participants (mean age 71.9+/-8.6 years, ModEl group) and 9 young physically active participants (mean age 25.8+/-2.3 years, AcYo) participated in this study. Concentrations of plasma ionised calcium (iCa), serum parathyroid hormone (iPTH), 25-hydroxy-vitamin D [25(OH)D], and 1,25-dihydroxy-vitamin D3 [1,25(OH)(2)D3] as well as the bone biochemical markers type-I collagen C-telopeptide (CTX) for bone resorption and osteocalcin (OC) and bone alkaline phosphatase (B-ALP) for bone formation, were analyzed before and after a maximal incremental exercise test. In all groups, iCa decreased significantly (p<0.05 for ModEl and AcYo and p<0.001 for HAcEl) while iPTH increased significantly (p<0.01 for ModEl and HAcEl and p<0.001 for AcYo) after exercise. The levels of 1,25(OH)(2)D3, OC and CTX remained unchanged, while 25(OH)D decreased only in HAcEl group while B-ALP increased in ModEl group. In conclusion, strenuous exercise disturbed calcium homeostasis, mainly the iCa/iPTH equilibrium independently of gender, age or fitness level of the participants while no immediate effect on bone turnover was observed.