ISO 14243 is the governing standard for wear testing of knee prostheses, but there is controversy over the correct direction of anterior-posterior (AP) displacement and loading and the correct direction of tibial rotation (TR) angles and torque. This study aimed to analyze how altering the direction of AP and TR affected wear on the tibial insert. Modifications to the conditions specified in ISO 14243-1 and ISO 14243-3 were also proposed. As such, five loading conditions were applied to FEA models of a knee prosthesis: (1) Modified ISO 14243-3 with positive AP displacement and TR angle, (2) ISO 14243-3:2004 with negative AP displacement and positive TR angle, (3) ISO 14243-3:2014 with positive AP displacement and negative TR angle, (4) Modified ISO 14243-1 with positive AP load and TR torque, and (5) ISO 14243-1:2009 with negative AP load and positive TR torque. This study found that changing the input directions for AP and TR according to ISO 14243-1 and 14243-3 had an influence on the wear rate and wear contours on the tibial insert model. However, the extent of wear varies depending on the design features of the tibial insert and shape of the input curves. For displacement control according to ISO 14243-3, changing the direction of AP displacement had a marked influence on the wear rate (272.77%), but changing the direction of TR angle had a much lower impact (2.17%). For load control according to ISO 14243-1, reversing the AP load (ISO 14243-1:2009) only increased the wear rate by 6.73% in comparison to the modified ISO 14243-1 conditions. The clinical relevance of this study is that the results demonstrate that tibial wear is affected by the direction of application of AP and TR. Incorrect application of the loading conditions during the design stage may lead to an ineffective preclinical evaluation and could subsequently influence implant longevity in clinical use.