In ancient buildings, timber members may require specific on-site interventions, including reinforcement or repair and sometimes the insertion of reinforcing material into grooves routed in the original sound wood. The required number, positions, and dimensions of grooves and the strengthening materials may differ according to the desired increases in bending stiffness and strength. The modulus of elasticity (MOE) of each beam is of key importance: the MOE is typically a point of weakness for wood and is considered a constant characteristic of each beam. However, taking into account the wood lost for the groove, whether the needed incisions affect the stiffness is unknown. As such, in this study, 12 old beams were accurately measured, and their static and dynamic MOEs were calculated before and after groove formation to simulate the typical processes of reinforcing interventions. One groove was routed along the length of each beam and progressively deepened in three steps. The results of tests showed how the MOE is affected by the groove depth, decreasing by up to one-third (34.1%), and that beam stiffness cannot be regarded as constant. Beam stiffness depends on the features of the beam, mainly the pith, fissures, and slope of the grain, as well as its structural integrity. Beam stiffness is strongly influenced by the cuts upon it. This study proves how the weakening effect of grooves created on-site can be assessed using the dynamic MOE and roughly predicted with a visual survey. The grooves required for strengthening beams must be thoroughly evaluated, considering the potential reduction in the mechanical properties of the beam, which goes well beyond the wood lost during cutting.