Previous studies suggest that seasoned cyclists may incur a low bone mineral density. This study investigated the effect of multiday cycling on bone turnover. Ten male cyclists completed 4 consecutive days of cycling for 3 h·day(-1). Sweat calcium excretion during exercise and serum calcium, cortisol, bone formation marker (bone alkaline phosphotase (bone-ALP)), bone resorptive marker (C-terminal telopeptide of type I collagen (β-CTX)), and parathyroid hormone concentration were measured before and immediately postexercise each day. Serum β-CTX concentration increased from pre- to postcycling on days 1 and 2 (p = 0.01) (day 1: 0.31 ± 0.14 to 0.60 ± 0.4 ng·mL(-1); day 2: 0.58 ± 0.26 to 0.87 ± 0.42 ng·mL(-1)), while serum bone-ALP concentration remained unchanged. Conversely, on days 3 and 4 both serum β-CTX (day 3: 0.60 ± 0.26 to 0.43 ± 0.26 ng·mL(-1), p < 0.05; day 4: 0.63 ± 0.21 to 0.43 ± 0.22 ng·mL(-1), p < 0.001) and bone-ALP (p < 0.01) response to exercise was suppressed. Interestingly, calcium lost to sweat and postexercise serum cortisol concentration were also significantly lower on days 3 and 4 than on day 1 (p < 0.05). However, both serum β-CTX (102%-124%) and bone-ALP (25%-29%) remained persistently elevated after 21 h of overnight recovery on all successive days compared with day 1 pre-exercise, where the percentage increase was greater for β-CTX (p < 0.05). Bone resorption, immediately following prolonged cycling, is acutely reduced by the third and fourth consecutive days and is coincident to reduced sweat calcium excretion and cortisol concentration. However, multiday cycling imposes a persistent increase in bone resorption following overnight recovery.