Skin blood flow (SkBF) measurements with laser Doppler probe provide a continuous real‐time index for local microcirculatory blood flow under the probe. However, because the area detected by a single probe is very small, it is known that SkBF reading may vary significantly if the position of the probe is moved slightly. The possible low reliability after replacing the probe limits the application of this measurement in some experiments (e.g. prior and after outdoor exercise). In this study, we tested the hypothesis that using multiple laser Doppler probes could increase the reliability of the measurements. SkBF, local skin temperature (Tsk) and blood pressure were measured with 3 repeated trials in 11 subjects (6 M, 5 F, 42 ± 5 yrs) in a temperature controlled room. Seven laser Doppler probes were placed in a custom‐made holder (round shape, diameter 45 mm). Trial 1: after the probe holder was placed on a forearm, the position was marked. After ~5 min rest, SkBF and Tsk (under the holder) were recorded for 3 min. Then, the probe holder was removed from the forearm and placed on the side calf of a leg to measure leg SkBF. After dis‐instrumentation, subjects were released from the testing table for 10–15 min. Trial 2 and 3: the probe holder was carefully replaced as close as possible on the same positions according to the markers on the skin. The measurements on forearm and then leg as well as the intervals between the trials were repeated. In this Test‐Retest Reliability study, two‐way mixed‐effects models with absolute agreement were used to compute the intraclass correlation coefficient (ICC). There was no significant difference in blood pressure and Tsk between the trials. With the measurements from each single probe, the lowest ICCs were 0.68 (95% Confidence Interval: 0.35, 0.89) for SkBF and 0.76 (0.48, 0.92) for cutaneous vascular conductance (CVC) at leg. With the averaged values from 7 probes, the ICCs were 0.86 (0.62, 0.96) for SkBF and 0.91 (0.74, 0.97) for CVC at leg, and 0.95 (0.87, 0.99) for SkBF and 0.96 (0.88, 0.99) for CVC at forearm. Moreover, with the averaged values from 4 contiguous probes, the ICCs were greater than 0.84. These data suggest that SkBF measurement with multiple laser Doppler probes in a custom‐made holder has high reliability after replacing the probes on the same subjects.Support or Funding InformationSupported by NIH MPI R01 HL141198 (Li and Cui) and UL1 TR002014 (Sinoway)