Soft biological tissues have a hierarchical architecture from the molecular to the macroscale, with structure–function relations at each level crucial for function. In developing new soft biomaterials for medical applications, understanding, and emulating these mechanisms will provide essential guidance. In this chapter we review how time- and position-resolved synchrotron small-angle X-ray scattering (SAXS) combined with multiscale mechanical modelling can illuminate such small-scale mechanisms, using the examples of articular cartilage and the mutable connective tissue of echinoderms. In articular cartilage, SAXS reveals a gradient in fibrillar-level pre-strain, which is suppressed either by physiological static loading or by enzymatic modifications mimicking ageing, and modelling of the fibril/proteoglycan network shows that the pre-strain reflects the local internal swelling pressure. In mutable connective tissue, our results show that interfibrillar stiffening and de-stiffening enable its rapid alterations in mechanical properties, whose kinetics can be captured by analytical modelling of the structure. The combination of multiscale modelling and in situ SAXS thus shows potential in investigating and elucidating the mechanisms enabling function in both natural tissues as well as in new soft biomaterials mimicking their structure.