Fiber optic sensors are emerging as effective alternatives for the health monitoring of civil structures. Distinctive advantages over conventional electronic sensors are the immunity to electromagnetic interferences, the dielectric performances, the high survivability in aggressive environments and the high degree of miniaturization. Single-mode fiber optic sensors based on spontaneous Brillouin scattering add the unique feature of measuring distributed strain and temperature profiles along structural members. Measurement is based on the correlation of strain and temperature with the frequency shift of the Brillouin backscattered light induced by a pump light pulse launched into an optical fiber. The technique has significant potential for the structural health monitoring of bridges and in perspective, of integrated transportation infrastructures. However, very few field applications have demonstrated its feasibility on large-scale structures. The paper presents a pilot application of Brillouin Optical Time Domain Reflectometry (BOTDR) for the extensive strain measurement along four steel girders of a major highway bridge located in Osage Beach, Missouri, USA. Bridge A6358 is built with five continuous symmetrical spans: the two external are 44.8 m and 56.4 m long, respectively, while the central one has a length of 61 m, resulting in a total bridge length of 263.4 m. Each internal support consists of reinforced concrete (RC) bents supported by two RC circular piers having a 1.8 m diameter. The cross section comprises five composite, equally spaced, steel I-girders acting compositely with a 216 mm thick RC deck, with an out-to-out deck and clear roadway width of 12.4 m and 11.6 m, respectively. Measurements were made during a diagnostic load test that was conducted to assess the safety of the newly constructed bridge while acquiring the distributed girder strains under controlled load conditions. In addition, deflections at discrete locations were measured using a high-precision, non-contact automated total station (ATS) system that was previously evaluated with respect to Linear Variable Differential Transformers (LVDTs) in similar applications. A 1159 m long optical circuit was installed on the web of four girders at different depths, along up to two continuous spans, using an on-purpose designed aluminum/FRP inspection cart moving along the bottom flanges of two adjacent I-girders. The sensing circuit comprised bare optical cables and a custom-made "smart" glass Fiber Reinforced Polymer (FRP) tape with embedded optical sensors for strain measurement and thermal compensation. The specific correlation coefficients between the Brillouin frequency shift and the strain/temperature in each fiber type were preliminarly evaluated in the laboratory. The optical attenuation of the BOTDR circuit was kept within a value of 6 dB in the first 1026 m, thereby enabling to measure strains with a minimum accuracy of ±40 με on a length resolution of 2 m, using a AQ8603 optical strain analyzer unit set for 20 ns laser pulses. During the diagnostic load test, the data acquired from the AQ8603 unit were processed in real-time through a proprietary software in order to compensate for thermal deformations. Three-dimensional linear-elastic finite element analysis was performed to estimate the theoretical response of each girder. The numerical results approximate the ideal bridge response assuming full contribution of secondary structural members to the transverse load distribution, thus resulting in lower bound strain profiles. Hence, the FEM was calibrated based on the results of ATS deflection measurement in order to provide a reliable benchmark to evaluate the performance of the BOTDR system. Upper bound strain profiles were also computed via one-dimensional beam analysis as per the AASHTO LRFD Bridge Design Specifications. Despite the non ideal working conditions during circuit installation, which may affect the measurement quality, the experimental strain profiles accurately described the global bridge response under typical load test conditions. In case of steep geometrical discontinuities, a higher degree of accuracy was observed at strain levels above 100 με, which may suggest the use of pre-straining fixtures. The system performance was poor when bare fibers with several sharp bends had to be used, and in the circuit portion where a significant optical attenuation was detected. The outcomes of this pilot project demonstrate the practical potential of BOTDR sensors systems for the distributed strain measurement in large-scale bridge structures, and support the need of further research to improve and refine the technology, as well as reduce strain analyzer and specific equipment costs.