Purpose <p>Aim was to investigate relationship of gender and training experience with post-occlusive hyperemia (PORH) and answer how differences between men and women and their training status led to differences in vascular responses after occlusions.</p> Methods <p>This prospective experimental study included 80 healthy volunteers divided into four groups of 20: trained men, trained women, non-training women, and non-training men. The occlusion intervention on the dominant thigh was performed. The laser Doppler flowmetry was used to assess post-occlusion responses by measuring: arterial occlusion pressure—AOPmin[mmHg], total arterial occlusion pressure—AOP100%[mmHg], resting flow—RF[PU], time to peak—TP[s], recovery time—TR[min], and biological zero—BZ[PU].</p> Results <p>Men recovered faster than women in trained and untrained groups and showed higher AOPmin, and AOP100% compared to women in both trained and untrained groups. Women had higher RF comparing to men in both trained and untrained groups, and higher BZ but only in the untrained group. Trained people had higher RF, AOPmin and recovered faster to baseline, but had lower TP than non-trained. Trained men had higher AOP100% than untrained men; in women, it was non-significant. There were no significant differences between genders in TP in both trained and untrained subgroups, and in BZ in trained subgroups.</p> Conclusions <p>There are significant differences between trained and untrained men and women in microcirculatory parameters after occlusion intervention. Between gender differences were noted only in some parameters which underscores the need of further research.</p>