Serum microfibrillar-associated protein 4 (sMFAP4) has been investigated as a biomarker for various diseases and is demonstrated to show significant gradual increase with severity of liver fibrosis. Ideal biomarkers used for disease diagnosis or prognosis should display deviating levels in affected individuals only and be robust to factors unrelated to the disease. Here we show the impact of normal physiological variation of sMFAP4 by characterizing the circadian variation, week-to-week variation, and physical exercise-induced levels. Serum samples from 3 groups of healthy volunteers were drawn: 7 times during a 24-hour period, 5 times during a 3-week period, and before and after a standardized physical exercise challenge. sMFAP4 was determined by AlphaLISA. Statistical analysis was performed using mixed effects modeling of repeated measurements. Circadian variation of sMFAP4 was demonstrated, with time of peak and nadir values depending on age and gender. For males, the peak values were observed during nighttime whereas for females, peak values were observed in the morning. Individual sMFAP4 levels remained stable over a period of 3 weeks and physical exercise inferred a mild negative influence. In conclusion, the circadian sMFAP4 variation was significant, and the levels could be influenced by physical activity. However, these variations were of limited magnitude relative to previously observed disease-induced levels in support of the biomarker potential of sMFAP4.