Introduction 6 13 17 3 4 12 22 24 Therefore, the purpose of this study was to assess the applicability and reproducibility of VOP for blood flow measurements in the calf (CBF) during HUT at different tilt angles (0°, 30°, 45°, and 70°). In a subgroup CBF measurements by VOP were compared with blood flow measurements using Doppler ultrasound. To assess reproducibility of VOP, measurements were performed twice. Materials and methods Subjects In total eighteen healthy, normotensive subjects aged 21–30 years volunteered to participate in this study. In eight subjects blood flow measurements using VOP were compared with blood flow measurements using Doppler ultrasound (DU). In nine other subjects the VOP measurements were repeated within two weeks to assess reproducibility. 1 Table 1 Subject characteristics  Mean ± SD Age, years 25 ± 4 Length, cm 187 ± 9 Body mass, kg 80 ± 11 Calf circumference, cm 38 ± 2 Systolic blood pressure, mmHg 125 ± 12 Diastolic blood pressure, mmHg 74 ± 5 Heart rate, bpm 64 ± 10 Measurements 8 9 Protocol Supine blood flow measurements using DU and VOP were performed after subjects were 30 minutes quietly in supine position. When the subject was 2 minutes into 30° HUT, DU measurements started until 3.5 minutes where after CBF continued for another 3.5 minutes. The venous occlusion pressure was adjusted to the hydrostatic pressure column, which is derived from the vertical distance heart level–thigh level and was calculated as the sinus of the tilt angle * actual distance heart–thigh, and was 75 mmHg during 30° HUT. The same procedure was repeated for 45°, and 70° HUT using a venous occlusion pressure of 87, and 105 mmHg, respectively. Data analysis −1 −1 25 8 −1 −1 Statistics Data are expressed as mean ± standard deviation (SD). 5 t 25 t P Results In five volunteers, CBF could not be measured during 70° head-up tilt (HUT), due to a poor plethysmography signal or near fainting of the subject. 1 1 P Fig. 1 P    Agreement VOP and DU  P 2 3 Fig. 2 Blood flow in the superficial femoral artery (BF SFA) measured by Doppler ultraound during different angles of head-up tilt versus calf blood flow (CBF) measured by venous occlusion plethysmography corrected for lower leg volume. Pearson correlation coefficient is 0.86  Fig. 3 Relative difference between the blood flow measured by venous occlusion plethysmography (VOP) and the superficial femoral artery blood flow measured by Doppler ultrasound (DU) versus the mean of both flow for each individual subject at different tilt angles  Reproducibility of VOP during head-up tilt 2 Table 2 n Subject Supine 30° HUT 45° HUT n test 1 test 2 test 1 test 2 test 1 test 2 test 1 test 2 Mean ± SD 2.6 ± 0.6 2.6 ± 0.8 1.3 ± 0.3 1.3 ± 0.3  1.1 ± 0.3 1.1 ± 0.2 n n %change ± SD   −45 ± 11 −49 ± 12 −54 ± 11 −57 ± 13 −44 ± 17 −44 ± 14 CV 15.0%(CI 10.1–29.1) 11.0%(CI 7.4–21.3) 14.9%(CI 10.0–28.9) 8.7%(CI 4.9–33.2) −1 −1 Discussion Calf blood flow (CBF) measured with VOP correlates well with superficial femoral artery blood flow (BF SFA) measured with DU, and can be measured reproducibly during HUT. Since the most profound changes in blood flow with both techniques were already measured in 30° HUT, and the increase in hydrostatic and venous pressure, and concomitant technical difficulties are smallest from supine to 30° HUT we recommend to use VOP for leg blood flow measurements during HUT up to 30°. 2 7 10 11 14 16 27 21 28 1 18 25 15 19 20 26 4B 5 Fig. 4 A B A B  Fig. 5 Typical plethysmographic tracing of one individual subject during a complete experiment. Blood flow measurements at 30° HUT start when the plethysmography signal does not change anymore, meaning that venous volume reached a steady state situation. Besides, looking at a typical VOP tracing at 30° HUT, the increase in venous volume is linear during the first 5 seconds of cuff inflation, indicating that blood flow measurements using VOP during 30° HUT are not compromised by a decrease in venous compliance  Limitation Using VOP, blood flow is defined as limb volume changes over time. During HUT, when the leg is below heart level, volume changes can still be measured using VOP, however, the physiological determinants of these volume changes are complex and it is no longer possible to say with reasonable certainty that a change in volume over time, which most likely reflects flow, is determined by resistance vessel tone. For example, limb blood flow measured using VOP in HUT position can decrease due to an increase in venous pressure, as a result of venous congestion and the associated fall in arterio-venous pressure gradient, without any increase in resistance at the arteriolar level. In conclusion, this study demonstrates that CBF measured by VOP during HUT is suitable and reproducible. The method is easy applicable and recommended in tilt angles equal to 30° to avoid high hydrostatic and leg venous pressures.