This study assesses the variability of two non-invasive methods of measuring stratum corneum barrier function in vivo. Transepidermal water loss (TEWL), and the vascular response to hexyl nicotinate (HN) penetration as determined by laser-Doppler flowmetry, were measured in a group of 21 healthy volunteers. Each time profile of the vascular response to HN penetration was analysed using the following parameters: the baseline cutaneous blood flow, the lag-time between application and initial response (to), the time between application and maximum response (tmax.), the maximum response, and the slope of the curve. TEWL measured on the left volar forearm showed a normal range of 3.9–7.6 g/m2h and a small inter-individual variability [coefficient of variation (CV) 19–4%]. TEWL values at three other forearm sites did not show differences of clinical importance compared with the left volar forearm. The parameters of the vascular response to HN penetration spanned a wider normal range than the TEWL values (CV between 33 and 52%). Repeat measurements after a 1–2 month interval showed highly reproducible individual TEWL values. The mean difference between first and second measurements was only 0.03 g/m2h: the relative difference 0.6%. The intra-individual reproducibility of to and tmax for HN penetration was also high (relative differences of 2.8 and 3.1%, respectively). The other vascular response parameters were less reproducible (relative differences of 6.9–18.6%). We conclude that TEWL and selected parameters of HN penetration, as non-invasive tests of the stratum corneum barrier function, yield reproducible results and are hence useful for investigations assessing the skin barrier function in various disorders.