Alpha-keratin of nails is formed of fibrillar keratins and globular keratin associated proteins. The diagnosis of systemic diseases and forensic argumentation from fingernails primarily requires a comprehensive examination of the α-keratin composition and structure, including their gender dependence in healthy subjects. Nail clippings were collected from 78 healthy volunteers (46 females and 32 males; mean age of 22.3 ± 3.5 years). The snippets were cleaned, pulverized, lyophilized and studied using solid-state ¹³C CP/MAS NMR. The NMR spectra were deconvoluted into separate signals, the areas of which were then subjected to statistical analyses: cluster analysis, principal component analysis and tests for unpaired groups. With regard to the amino acid composition of the nail proteins, males have more Leu and Ala, and less Cys, Arg and Ile, than females. Furthermore, the α-keratin of males has more disulphide bonds, and more β-sheet and random coil secondary structures, but fewer α-helical regions. The higher content of disulphide bonds is possible in the α-keratin of males despite the slightly lower Cys content and it renders their fingernail plates stiffer than those of females.