## Discussion
However, this leads to a limitation of the study, that is, the use of a relatively small homogenous sample. Despite this, 10 data points (ROIs) were taken from each elastogram at each testing session and there were three testing sessions in the time of day section (08:00 h, 12:00 h and 17:00 h) and six in the exercise section. Therefore, the total numbers of measures taken from all participants over the course of the testing and included in the analysis for this study is over 1500 measures, a number much higher than that used in previous research. All participants were considered healthy and free of lower limb injury. This was considered important as until the normal variations within healthy tendons are established and some baseline clinical values recorded, it will be impossible to establish what is normal, acceptable change, versus pathological change and therefore, of concern. It is however not possible to generalize the results from this study to any symptomatic individuals with pathology or who fall outside the tested age range. Future research should define other populations based on age, sex or other covariates expected to influence stiffness as well as examining the results obtained from pathological samples. It is worth noting here the limitations of SWE with regards to anisotropy, as SWE utilises ultrasound to trace the propagation of generated shear waves, so with ultrasound scanning, a controlled angle of the transducer to the skin is crucial in obtaining an accurate image and obtaining reliable values for SWE. Shear waves propagate more readily along longitudinal fibres than they do across them [36] and therefore it has been noted that the transducer for SWE should be orientated parallel to the direction of the fibres being assessed to ensure the most accurate results [53]. Our own previous work [54] also demonstrated that certain protocols can be utilised when using SWE to assess the stiffness of the human Achilles tendon in vivo to standardise the measurements and obtain the most accurate results. These include using longitudinal images and keeping the foot in a relaxed position whilst scanning. The exercise mode utilised in this study was running, however other modes of exercise such as stretching have been shown to also significantly alter tendon stiffness over short time periods [13]. It would be of great interest to look at the impact other types of exercise have on the SWV in the AT in comparison to those found with running. Lastly, whilst the results of this study and other similar research [25] would indicate that the running-induced increase in AT stiffness is a temporary effect, other research does link long term adaptation of tendons to long term exercise training. There are currently no studies that trace the stiffness of an AT from sedentary to long term duration running, and none that have studied the AT stiffness of a long-term runner who stops training and therefore future research could elaborate on this. In conclusion, this is the first study to utilise SWE to assess differences in leg dominance, trace the stiffness of the human AT in vivo during a normal working day and assess the impact of an acute bout of running. The results indicate that leg dominance does not affect SWE results in asymptomatic, healthy tendons, therefore the contra-lateral tendon may be used as a comparison for clinical investigation for this population. The time of day that a SWE measure is taken does not significantly alter AT stiffness and does not influence the measured values by more than 2.07%. Therefore, clinical appointments can be scheduled between 08:00 h and 17:00 h without affecting the measures taken, improving appointment accessibility when using SWE. Lastly, this study has shown that a 30-min run has no impact on AT length, however it does result in significant decreases in max AP diameter and significant increases in SWV measures, a surrogate measure of stiffness. The measured max AP diameter and SWV measures return to PRE-like values when measured 6 h after the exercise.