This study aimed to investigate the preferential collagen fibril alignment in unloaded and loaded tendons using elastic scattering spectroscopy. The device consisted of an optical probe, a pulsed light source (320-860 nm), a spectrometer and a PC. Two probes with either 2.75-mm or 300-µm source-detector separations were used to monitor deep and superficial layers respectively. Equine superficial digital flexor tendons were subjected to ex vivo progressive tensional loading. Seven times more backscattered light was detected parallel rather than perpendicular to the tendon axis with the 2.75-mm separation probe in unloaded tendons. In contrast, using the 300-µm separation probe the plane of maximum backscatter (3-fold greater) was perpendicular to the tendon axis. There was no optical anisotropy in the cross-sectional plane of the tendon (i.e. the transversely cut tendon surface), with no structural anisotropy. During mechanical loading (9- % strain) backscatter anisotropy increased 8.5-8.5-old along the principal strain axis for 2.75-m probe separation, but almost disappeared in the perpendicular plane (measured using the 300-[mu]m probe separation). Optical (anisotropy) and mechanical (strain) measurements were highly correlated. We conclude that spatial anisotropy of backscattered light can be used for quantitative monitoring of collagen fibril alignment and tissue reorganisation during loading, with the potential for minimally invasive real-time structural monitoring of fibrous tissues in norm, pathology or under repair and in tissue engineering.