Background: Direct assessment of the effect of postural changes on interstitial fluid pressure (IFP) in the human skin under physiological conditions is important for the understanding of mechanisms involved in diseases resulting in lower limb edema. Previous techniques to measure IFP had limitations of being invasive, and acute measurements were not possible. Here we describe the effect of postural changes on IFP in the skin of the foot using the minimally invasive servonulling technique. Results: Measurements were performed in 12 healthy subjects. IFP (means ± SD) was significantly higher in the sitting (5.1 ± 2.9 mm Hg) than in the supine position (–0.3 ± 3.6 mm Hg, p = 0.04) when measured in the sitting position first. The difference between the sitting and the supine position was not significant when measurements were taken in the supine position first [from 1.0 ± 4.3 (supine) to 3.6 ± 6.7 mm Hg (sitting), p = 0.46]. Spontaneous low-frequency pressure fluctuations occurred in 58% of the recordings during sitting, which was almost twice as frequent as in the supine position (33%; p = 0.001), while no effects on lymphatic capillary network extension were observed (p = 0.12). Conclusion: Using the servonulling micropressure system, postural effects on IFP can be directly assessed. IFP is higher in the sitting position, but differences are influenced by the time in the upright position.