Locomotion is one of the most important abilities of humans. Actually, gait locomotion provides mobility, and symbolizes freedom and independence. However, gait can be affected by several pathologies, due to aging, neurodegenerative disease, or trauma. The evaluation and treatment of mobility diseases thus requires clinical gait assessment, which is commonly done by using either qualitative analysis based on subjective observations and questionnaires, or expensive analysis established in complex motion laboratories settings. This thesis presents a new wearable system and algorithmic methods for gait assessment in natural conditions, addressing the limitations of existing methods. The proposed system provides quantitative assessment of gait performance through simple and precise outcome measures. The system includes wireless inertial sensors worn on the foot, that record data unobtrusively over long periods of time without interfering with subject's walking. Signal processing algorithms are presented for the automatic calibration and online virtual alignment of sensor signals, the detection of temporal parameters and gait phases, and the estimation of 3D foot kinematics during gait based on fusion methods and biomechanical assumptions. The resulting 3D foot trajectory during one gait cycle is defined as Foot Signature, by analogy with hand-written signature. Spatio-temporal parameters of interest in clinical assessment are derived from foot signature, including commonly parameters, such as stride velocity and gait cycle time, as well as original parameters describing inner-stance phases of gait, foot clearance, and turning. Algorithms based on expert and machine learning methods have been also adapted and implemented in real-time to provide input features to recognize locomotion activities including level walking, stairs, and ramp locomotion. Technical validation of the presented methods against gold standard systems was carried out using experimental protocols on subjects with normal and abnormal gait. Temporal aspects and quantitative estimation of foot-flat were evaluated against pressure insoles in subjects with ankle treatments during long-term gait. Furthermore, spatial parameters and foot clearance were compared in young and elderly persons to data obtained from an optical motion capture system during forward gait trials at various speeds. Finally, turning was evaluated in children with cerebral palsy and people with Parkinson's disease against optical motion capture data captured during timed up and go and figure-of-8 tests. Overall, the results demonstrated that the presently proposed system and methods were precise and accurate, and showed agreement with reference systems as well as with clinical evaluations of subjects' mobility disease using classical scores. Currently, no other methods based on wearable sensors have been validated with such precision to measure foot signature and subsequent parameters during unconstrained walking. Finally, we have used the proposed system in a large-scale clinical application involving more than 1800 subjects from age 7 to 77. This analysis provides reference data of common and original gait parameters, as well as their relationship with walking speed, and allows comparisons between different groups of subjects with normal and abnormal gait. Since the presented methods can be used with any foot-worn inertial sensors, or even combined with other systems, we believe our work to open the door to objective and quantitative routine gait evaluations in clinical settings for supporting diagnosis. Furthermore, the present studies have high potential for further research related to rehabilitation based on real-time devices, the investigation of new parameters' significance and their association with various mobility diseases, as well as for the evaluation of clinical interventions.