Many biomechanical studies are performed on dense connective tissues in the laboratory over a substantial time period; however, the effect of the in vitro testing environment on the cyclic load-relaxation behaviour of these structures is not well established. This study evaluated the effect of long duration of testing on ligament viscoelastic behaviour, using the rabbit femur-medial collateral ligament-tibia complex as a model. Dissected rabbit knee joints were mounted on a materials testing machine, and isolated ligament complexes were cycled at a frequency of one cycle/min to a fixed displacement of 0.7 mm for an 18-hour period. After an initial period of exponential load relaxation, the cyclic peak loads slowly decreased over the 18-hour period. The average decrease in the cyclic peak load between 0.5 and 18.0 hours was 0.26% (of the original peak load) per hour (r2 = 0.934), or a total of 8.6+/-4.6% over this period (p < 0.0001). Thus, low-load testing of dense connective tissues in the laboratory over extended periods significantly alters their biomechanical behaviour, and these changes should be considered in long-term laboratory-based studies of dense connective tissues.