Wear testing cannot replicate the variations in wear rates and wear mechanisms seen in vivo, which may be related to differences between in vivo and in vitro conditions. A considerable difference exists between the in vivo synovial fluid volume (few milliliter) and the in vitro substituted bovine serum volume (several hundred milliliter). The aim of this study was to analyze the effects of a reduced fluid volume on the wear behavior in a knee wear simulator study. Four wear tests with decreasing fluid volumes (250, 150, 75, and 45 ml) were carried out. Using a large fluid volume of 250 ml for wear testing resulted in a wear rate of 9.7 ± 1.2 mm3/106 cycles. Decreasing the fluid volume consecutively reduced the wear rate to down to 8.8 ± 1.4 mm3/106 for 150 ml (p = 1.00), 5.6 ± 1.2 mm3/106 for 75 ml (p = 0.01), and 1.0 ± 0.2 mm3/106 cycles for 45 ml fluid volume (p ≤ 0.01). Additionally, higher serum degradation and larger wear particles were observed with smaller fluid volumes used for testing. This study demonstrates the high relevance of the protein-based lubricant on the wear behavior and the technical limitation to replicate the synovial fluid in simulator tests. Wear testing should be carried out using larger fluid volumes (e.g., 250 ml) to generate physiological relevant wear masses. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:92–97, 2015.