This paper presents the design, modeling, analysis, fabrication, and experimental characterization of the soft robotic ankle-foot orthosis (SR-AFO), which is a wearable soft robot designed for ankle assistance, and a pilot human study of its use. Using two novel pneumatically powered soft actuators, the SR-AFO is designed to assist the ankle in multiple degrees-of-freedom during standing and walking tasks. The flat fabric pneumatic artificial muscle (ff-PAM) contracts upon pressurization and assists ankle plantarflexion in the sagittal plane. The multi-material actuator for variable stiffness (MAVS) aids in supporting ankle inversion/eversion in the frontal plane. Analytical models of the ff-PAM and MAVS were created to understand how the changing of the design parameters affects tensile force generation and stiffness support, respectively. The models were validated by both finite element analysis and experimental characterization using a universal testing machine. A set of human experiments was performed with able-bodied participants to evaluate: (a) lateral ankle support during quiet standing, (b) lateral ankle support during walking over compliant surfaces, and (c) plantarflexion assistance during push-off in treadmill walking. Group results revealed increased lateral ankle stiffness during quiet standing with the MAVS active, reduced lateral ankle deflection while walking over compliant surfaces with the MAVS active, and reduced muscle effort in ankle platarflexors during 40–60% of the gait cycle with the dual ff-PAM active. The SR-AFO shows promising results in providing lateral ankle support and plantarflexion assistance with able-bodied participants, which suggests a potential to help restore the gait of impaired users in future trials.