Purpose This aim of this study was to assess the possible association between diurnal oscillations and biochemical markers associated with calcium homeostasis. This included the markers parathyroid hormone (PTH), total calcium, total alkaline phosphatase, phosphate, and 25-hydroxyvitamin D (25-OH-D). By examining the influence of circadian rhythms on these parameters, the study aimed to deepen the understanding of calcium metabolism dynamics and its clinical implications. Patients and methods Blood samples from 24 Caucasian male volunteers aged 20 to 40 (mean age 26) with normal pulse, blood pressure, and BMI were analyzed for biochemical markers related to calcium homeostasis. Data was obtained from the Bispebjerg study of diurnal variations. Blood samples were collected every three hours over a 24-hour period. Patients were fasting from 22:00 to 09:00. The participants spent 24 h in the hospital ward, receiving regular meals and engaging in low-intensity activities. They experienced 15 h of daylight and 9 h of complete darkness during sleep. Diurnal oscillations were analyzed using cosinor analysis with statistical significance set at p < 0.05. Results Total calcium, phosphate, and PTH exhibited significant diurnal variations. Total calcium and PTH were inversely synchronized while PTH and phosphate oscillated in synchronization. The three parameters showed relatively large amplitude/reference range ratios from 25.4% to 41.5%. Conclusion This study found notable fluctuations in total calcium, phosphate, and PTH levels over a 24-hour cycle, while 25-OH-D and total alkaline phosphatase remained consistent. It highlights the importance of considering sampling times for total calcium, PTH, and phosphate in clinical settings.