Bone resorption can be evaluated by measuring the urinary excretion of collagen type I cross-linked N telopeptides (NTx). Since it is difficult to obtain (and verify) 24 h urine collections from patients, untimed spot urines are more practical. Such measurements, however, need correction for urine dilution and potentially may vary with collection time since a circadian rhythm in bone metabolism has been reported. This study examined cross-link excretion in urine voids serially collected during a 24 h period from subjects living their normal daily routine (as opposed to a controlled hospital setting). This mimics the situation for walk-in patients visiting a clinician and providing a spot urine. A total of 35 dentists (20 males, 15 females) collected all urine voids separately over a 24 h period. Urines were analyzed for creatinine and NTx. The effects of time of day on the excretion rates of these metabolites (in nmol/h) and on the cross-link:creatinine ratio were assessed. A circadian rhythm was evident in the excretion rate of creatinine with a peak in the late afternoon (18% higher than the 24 h mean, p = 0.0004). The NTx excretion rate peaked in the morning (9% higher than the 24 h mean) but this latter rhythm was not statistically significant (p = 0.31). The NTx:creatinine ratio fell during the day from a high (122% of the 24 h mean) in the early morning to a low in the early evening. This rhythm in the NTx:creatinine ratio in untimed spot urines was statistically significant (p < 0.0001). In conclusion, the NTx:creatinine ratio in spot urines from adult outpatient subjects showed a significant circadian rhythm. Variations in creatinine excretion were the primary cause. Time of day should, therefore, be taken into account when comparing test results of spot urines with normal ranges or with other samples from the same subject.