Peripheral sudomotor dysfunction is present in many peripheral neuropathies, but structural assessments of sudomotor fibers rarely occur. We evaluated 36 diabetic and 72 healthy control subjects who underwent detailed neurologic examinations and punch skin biopsies. Physical exam findings were quantified by neuropathy impairment score in the lower limb. Skin biopsies quantified intraepidermal nerve fiber density (IENFD) and sweat gland nerve fiber density (SGNFD) by a manual, automated, and semiquantitative method. The automated and manual SGNFD correlated with the IENFD at the same site (r = 0.62, P < 0.05 automated method, r = 0.67, P < 0.05 manual method). As neuropathy worsened, the SGNFD at the distal leg declined (automated counting r = -0.81, P < 0.001; manual counting r = -0.88, P < 0.001). The semiquantitative method displayed poor inter- and intrareviewer reliability and correlated poorly with standard neuropathy evaluation scores. Our results suggest that sudomotor fibers can be rapidly and reproducibly quantified, and results correlate well with physical exam findings.