Czekalla C, et al. – This research aspired to examine the effect of body site, age and gender on the collagen fibers, via noninvasive in vivo vertical two–photon microscopy. The highest second harmonic to autofluorescence aging index of dermis (SAAID) was estimated at the volar forearm as compared to the abdominal SAAID. It appeared to be considerably lower (p < 0.05). The gluteal region illustrated notably lowest SAAID (p < 0.05). The SAAID in female skin appeared to be higher compared to male skin and decreased with increasing age. These effects warranted advanced studies to be able to specifically detect and evaluate the influences.