Painted wooden panels are multi-layered structures composed of wood, animal glue, gesso and paints, in which the gesso layer is particularly vulnerable to humidity fluctuations due to the development of internal stresses and fracture. This study established experimentally a relationship between the strain magnitude leading to the first fracture of the gesso and the number of cycles at that strain, that is, the vulnerability of the gesso layer to fatigue damage. Specimens of single, wood panels coated with gesso were subjected to cycles of mechanical stretching and compression to imitate dimensional changes induced in the system by repetitive fluctuations of relative humidity (RH). The development of cracks in the gesso layer was monitored using a laser speckle decorrelation technique. Numerical modelling was used to calculate the amplitude of sinusoidal RH cycles which are allowable so as not to exceed the critical mismatch between the climate-induced responses of the unrestrained wood panel and the gesso layer respectively, which would lead to the first fracture of the virgin gesso after a defined period of cycle occurrence–100years in this study. The allowable amplitudes were derived as a function of cycle duration, panel thickness and the configuration of moisture exchange by a panel with the environment. The worst-case duration of the RH cycles, for which the allowable amplitude is at its minimum, were calculated for varying thicknesses of the panels. The analysis of the results revealed that the 10mm panel with two faces permeable to the water vapour flux and subjected to fluctuation cycles lasting 10days represents the ‘absolute’ worst case in the study performed, allowing only the fluctuation amplitude of ±14%.