Fluorescence spectroscopy is a promising technology for detection of epithelial precancers and cancers. While age and menopausal status influence measurements in the cervix, other variables do not significantly affect the diagnosis. In this study we examine probe pressure as a variable. A fiber optic probe to measure fluorescence spectra at different calibrated levels of pressure was designed and tested. A pilot study was conducted measuring fluorescence excitation emission matrices in 20 patients at light, medium, and firm pressure. Spectroscopic data were pre-processed and analyzed to compare mean peak intensities as a function of pressure. Further statistical analyses tested for differences in intensities at each excitation/emission wavelength pair. Four providers made measurements from 41 sites; 33 yielded good quality spectroscopic data (22 squamous normal, 7 squamous abnormal, 3 columnar normal, 1 transformation zone) from 17 of 20 patients. At all pressure levels, abnormal tissue showed less fluorescence intensity than normal tissue, and post-menopausal patients showed higher fluorescence intensity than premenopausal patients, consistent with previous analyses. A permutation analysis suggests that pressure does not significantly affect fluorescence intensity or lineshape. While other studies are needed to confirm these findings, this study suggests that fluorescence spectroscopy is a robust technology likely not influenced by fiber optic probe pressure. © 2004 Society of Photo-Optical Instrumentation Engineers.