Fast spreading antimicrobial resistance (AMR) is an emerging major public health issue. New therapeutic and diagnostic paradigms must be implemented to counteract this evolution. In this respect, fast antimicrobial susceptibility testing (AST) plays an important role by enabling optimum antibiotic prescriptions to improve the therapeutic efficiency on the one hand and to avoid excessive consumption of antibiotics on the other hand. Eventually, this could slow down AMR progression and extend the lifespan of current antibiotics. This thesis explores routes towards innovative fast AST technologies from 3 perspectives, using E. coli as a model organism: (i) microbial metabolic heat production, (ii) oxygen consumption assessment in response to antimicrobial exposure, and (iii) bacterial motility analysis after incubation without and with antibiotics. In the first project, we propose a chip-based isothermal nanocalorimeter, enabling reliable bacterial metabolic heat flow measurements. The platform’s high sensitivity enables bacterial growth detection within only a few hours of culture. Heat flow curves reflect growth rate and lag phase variations under different bacterial culture conditions and in the presence of antibiotics. At the first stage, for instance, we assessed bacterial heat flow in 3 different culture media, and we found that the overall heat production (i.e. heat flow integrated over time) depends on the availability of nutrients. As a proof-of-concept, we conducted a metabolic heat AST study based on 3 clinically relevant antibiotics featuring different functional mechanisms. The presence of antibiotics at sub-MICs (minimum inhibitory concentrations) leads to slower growth rates and lag phase elongation. In a parallel approach, we developed an on-chip bacterial oxygen consumption measurement system as an indirect calorimetric method. We focused on the aerobic metabolic activity of E. coli in the early phase of bacterial growth. Antibiotic exposure above MIC suppresses bacterial growth as well as oxygen consumption. In the third project, we further explored the properties of bacteria cultures facing antimicrobial stress. We performed an extensive motility study at single-bacterium resolution under different culture conditions. We developed a protocol using a composite microfluidic chip with UV-curable optical adhesive(OA)-patterned microchannels. The 4-ÎŒm high microfluidic channels confine bacterial suspensions in a quasi-2D space to facilitate high-resolution time-lapse imaging. In particular, we studied collective bacterial migration, appearing as "traveling bands" in the microchannel. For instance, we found that different inoculum sizes of isogenic bacteria result in heterogeneous swimming phenotypes. Interestingly, the collective migration properties were altered by the presence of antibiotics. We investigated this phenotypic diversity by quantifying bacterial motility based on parameters such as swimming velocity, tumble bias, and effective diffusion. In summary, the scope of this thesis was to take advantage of microfluidic technologies to assess the impact of antimicrobial exposure on E. coli from several aspects: metabolic heat flow, oxygen consumption, and swimming and motility behavior. The different platforms presented in this work are new tools enabling innovative protocols for fast in vitro diagnostics and general metabolism-related biological investigations.