The positions of Higher Order Laue Zone (HOLZ) Lines, visible in the bright field discs of Convergent Beam Electron Diffraction (CBED) patterns, are frequently used to determine the lattice parameters or strain present in phases observed in the Analytical Electron Microscope (AEM). The technique usually relies upon the use of a “standard” sample of known lattice parameter that has the same crystal structure as the unknown phase and is of similar average atomic number. The relative movement of particular HOLZ lines in the unknown phase with respect to the standard are used to calculate the difference in lattice parameters between the standard and the unknown. The thickness of the samples is not taken into consideration. This paper describes the results of both computer simulations and cold stage experiments in the AEM which lead us to conclude that the HOLZ lines are not independent of thickness, particularly in the presence of highly dynamical zero layer diffraction effects.