We examined variations of in vivo near-infrared Fourier transform Raman spectra on healthy human skin. Diurnal and day-to-day variation, variation between persons, variation between different spots in the same body region and variation in spectra obtained on the same spot was studied in 13 volunteers. In 140 volunteers we examined variations in Raman spectra caused by skin pigmentation. In our spectral analysis we concentrated on the wavenumbers and relative intensities of the water band around 3250 cm−1, the amide I and amide III bands in the regions around 1645–1680 and 1230–1300 cm−1, respectively, and on the wavenumbers of the strong bands at 1450 and 2940 cm−1 that were used as reference bands. The results showed a diurnal variation and interpersonal variation in relative water intensity. In relative amide I intensity there was a variation between persons and between repeated measurements on the same spot. Between persons the variation might arise from different hydration of proteins, but it is difficult to explain why this variation is seen when repeated measurements are made on the same spot. Day-to-day variations were seen in the relative amide III intensity. Variations in amide III band wavenumbers were seen between persons, probably because of different collagen structure. Pigmentation did not influence wavenumbers or intensities considerably, but a regression analysis showed a correlation between background height and pigmentation. The conclusion of our study is that Raman spectra obtained on healthy human skin are reproducible and show mostly small variations. Copyright © 2002 John Wiley & Sons, Ltd.