Blood glucose (BG) is usually measured after a caloric restriction of at least 8 h; however evidence-based recommendations for the duration of a fasting status are missing. Here we analyze the effect of fasting duration on levels of BG to determine the minimal fasting duration to achieve comparable BG levels to conventional fasting measurements. We used data of a cross-sectional study on primary care patients, performed in October 2005. We included 28,024 individuals (age-range 18-99 years; 63% women) without known diabetes mellitus and without missing data for BG and fasting status. We computed general linear models, adjusting for age, sex, time of blood withdrawal, systolic blood pressure, waist circumference, total- and HDL-cholesterol, physical activity, smoking, intake of beta-blocker and alcohol. We tested the intra-individual variability with respect to fasting status. Overall, the mean BG differed only slightly between individuals fasting ≥ 8 h and those fasting <8 h (men: 5.1 ± 0.8 mmol/L versus 5.2 ± 1.2 mmol/L; women: 4.9 ± 0.7 mmol/L, 5.0 ± 1.0 mmol/L). After 3 h of fasting differences of BG diminished in men to -0.08 mmol/L (95%-CI: -0.15; -0.01 mmol/L), in women to -0.07 mmol/L (-0.12; -0.03 mmol/L) compared to individuals fasting ≥ 8 h. Noteworthy, age, time of day of blood withdrawal, physical activity, and intake of hard liquor influenced BG levels considerably. Our data challenge the necessity for a fasting duration of ≥ 8 h when measuring blood glucose, suggesting a random sampling or a fasting duration of 3 h as sufficient. Rather, our study indicates that essentially more effort on the assessment of additional external/internal factors on BG levels is necessary.