This investigation proposes a synthetic biofluid, artificial sputum medium (ASM) and xanthan gum (XG), that mimics the mucus from a patient with cystic fibrosis, and investigates the rheological properties both macroscopically and microscopically. Macroscopic rheological characterization cannot address the heterogeneity or the behavior of particle transport inside the mucus. Microscopic rheology enables the characterization of the microenvironment by using microparticles as probes. The addition of XG to ASM provides a tunable parameter that enables the mechanical properties to be consistent with real mucus. Particles that were suspended in a media of ASM with XG displayed a subdiffusive behavior at short timescales with a diffusive exponent that decreases with an increase in concentration of XG. At long timescales, particles that were suspended in ASM+XG with a concentration of XG of 0.1% to 0.4% displayed diffusive behavior. While in more concentrated samples (0.5% and 1.0%), the particles were constrained inside local elastic “cages”. The microscopic moduli that were calculated showed consistently lower moduli than rotational rheometry. This discrepancy suggests that the solutions of XG have a hierarchical structure that better represents the weakly associated microstructure of mucus that is found in real sputum.