Purpose: X-ray cabinets are replacing 137 Cs/ 60 Co sources in radiation biology research due to advantages in size, handling, and radiation protection. However, because of their different physical properties, X-ray cabinets are more susceptible to experimental influences than conventional sources. The aim of this study was to examine the variations related to the experimental setups typically used to investigate biological radiation effects with X-ray cabinets. Materials and methods: A combined approach of physical dose measurements by thermoluminescence dosimetry and detection of biological effects by quantification of γH2AX and 53BP1 foci was used to analyze field inhomogeneity and evaluate the influence of the components of the experimental setup. Results: Irradiation was performed using an X-ray tube (195 kV,10 mA,0.5-mmthick copper filter, dose rate of 0.59 Gy/min). Thermoluminescence dosimetry revealed inhomogeneity and a dose decrease of up to 42.3% within the beam area (diameter 31.1 cm) compared to the dose at the center. This dose decrease was consistent with the observed decline in the number of radiation-induced foci by up to 55.9 %. Uniform dose distribution was measured after reducing the size of the radiation field (diameter 12.5 cm). However, when using 15-ml test tubes placed at different positions within this field, the dose decreased by up to 17% in comparison to the central position. Analysis of foci number revealed significant differences between the tubes for γH2AX (1 h) and 53BP1 (4 h) at different time points after irradiation. Neither removal of some tubes nor of the caps improved the dose decrease significantly. By contrast, when using 1.5-ml tubes, dose differences were less than 4%, and no significant differences in foci number were detected. Conclusion: X-ray cabinets are user-friendly irradiation units for investigating biological radiation effects. However, field inhomogeneities and experimental setup components considerably affect the delivered irradiation doses. For this reason, strict dosimetric monitoring of experimental irradiation setups is mandatory for reliable studies.