The recent growth of e-Commerce has induced an increasing freight demand, which could lead to negative externalities, in particular in urban areas. To foster sustainable development of cities and increase their livability, many local authorities are implementing urban vehicle access regulations, such as low-emission zones, banning the circulation of polluting vehicles. These measures prompted the adoption of new sustainable freight transport solutions for last mile deliveries, such as cargo bikes. The aim of this paper is to describe the study for the implementation of such a system. The procedure was tested (1) to define the location of a transshipment facility where parcels are moved from vans to cargo bikes, (2) to estimate the environmental and economic sustainability of the system, and (3) to quantify the effects of uncertainty in the final results. The framework was applied to the city center of Padova (Italy), where two sets of delivery system were considered: the first with traditional vans starting from an existing urban consolidation center and the second with manual and electric cargo bikes starting from a micro-depot. In particular, demand of home deliveries was estimated for a typical weekday; routes of freight transport means were defined by an optimization procedure; these data were used as input to a Discrete Event Simulation model. A sensitivity analysis was carried out modelling the potential uncertainty associated with load/unload times and travel speed of means, due to traffic congestion. Several scenarios were tested considering three locations as potential transshipment points. Outcomes of the simulations were used to estimate key performance indicators, evaluating the environmental and economic effects of the two delivery schemes. Results highlighted the potentiality of cargo bikes as a sustainable delivery system, and the impacts of uncertainty on the ranking of alternative options (i.e. micro-hubs).