Automated sorption balances are increasingly used to determine sorption isotherms of hygroscopic materials like wood. The sorption isotherms are constructed based on a number of equilibrium states between a specimen and the surrounding conditioning environment. In recent years, the uncertainty of these equilibrium states has been thoroughly discussed related to the time at constant environmental conditions needed to reach an adequate mass stability. However, uncertainties related to the conditioning environment are rarely discussed. This study describes a framework for analyzing and quantifying the effect of these uncertainties on the error in determined equilibrium moisture content. Various effects of the conditioning environment are analyzed, and the most important effects relate to the relative humidity (RH) in the instruments, both in terms of fluctuations in RH and an offset from the target value. The moisture error for these effects was found within the range 0.001–0.01 g g−1.