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Background Serum 25-hydroxyvitamin D (25(OH)D), a marker of vitamin D status influenced by sunlight exposure and diet, may affect various health conditions. This study investigates the tracking of serum 25(OH)D levels over 11 years in a general adult Norwegian population and identifies factors influencing future 25(OH)D levels.Methods Serum 25(OH)D was measured in 3511 adults participating in both second and third surveys of the Trøndelag Health Study (HUNT2: 1995–1997, HUNT3: 2006–2008). Serum 25(OH)D was measured using a chemiluminescent assay and calibrated for batch effect and seasonal variation. Tracking was evaluated using correlation coefficients, intraclass correlation coefficient (ICC), and coefficient of variation (CV). Multivariable linear regression examined relations between factors in HUNT2 and 25(OH)D in HUNT3.Results The correlation coefficient between 25(OH)D levels in HUNT2 and HUNT3 was 0.50 (95% CI: 0.47 to 0.52), with an ICC of 0.64 (95% CI: 0.56 to 0.70) and a CV of 18%. An average of 43% (range: 20.4%–54.9%) of participants remained in the same vitamin D category (<30.0, 30.0–49.9, 50.0–74.9 and ≥75.0 nmol/L) across both surveys. Male sex, vitamin D-decreasing polygenic score, higher body mass index and current smoking were associated with lower 25(OH)D levels, while higher education, moderate alcohol intake and vitamin D supplementation were associated with higher levels.Conclusions Serum 25(OH)D levels demonstrated moderate reliability over 11 years, suggesting that a single measurement may be insufficient for assessing long-term vitamin D status. Repeated measurements are recommended for accurate evaluation of vitamin D’s role in chronic disease risk.