Flexibility testing is one of the most important fitness assessments. It is generally evaluated by measuring the range of motion (RoM) of body segments around a joint center. This study presents a novel assessment of flexibility in the microcirculatory aspect. Eighteen college students were recruited for the flexibility assessment. The flexibility of the leg was defined according to the angle of active ankle dorsiflexion measured by goniometry. Six legs were excluded, and the remaining thirty legs were categorized into two groups, group H (n
= 15 with higher flexibility) and group L (n
= 15 with lower flexibility), according to their RoM. The microcirculatory signals of the gastrocnemius muscle on the belly were monitored by using Laser-Doppler Flowmetry (LDF) with a noninvasive skin probe. Three indices of nonpulsatile component (DC), pulsatile component (AC) and perfusion pulsatility (PP) were defined from the LDF signals after signal processing. The results revealed that both the DC and AC values of the group H that demonstrated higher stability underwent muscle stretching. In contrast, these indices of group L had interferences and became unstable during muscle stretching. The PP value of group H was a little higher than that of group L. These primary findings help us to understand the microcirculatory physiology of flexibility, and warrant further investigations for use of non-invasive LDF techniques in the assessment of flexibility.