Despite the known interplay between blood flow and function, there is currently no minimally invasive method to monitor diaphragm hemodynamics. We used contrast-enhanced ultrasound (CEUS) to quantify relative diaphragm blood flow (Q̇DIA) in humans and assessed the technique's efficacy and reliability during graded inspiratory pressure threshold loading. We hypothesized that 1) Q̇DIA would linearly increase with pressure generation, and 2) that there would be good test-retest reliability and inter-analyzer reproducibility. Can we validate the first minimally invasive method to measure relative diaphragm blood flow in humans? Quantitative CEUS of the costal diaphragm was performed in healthy participants (10M/6F; Age 28 ± 5 years; BMI 22.8 ± 2.0 kg·m-2) during unloaded breathing and three stages of loaded breathing on two separate days. Gastric and esophageal balloon catheters measured diaphragmatic pressure. Ultrasonography was performed during a constant-rate intravenous infusion of lipid-stabilized microbubbles after each stage. Ultrasound images were acquired after a destruction-replenishment sequence and diaphragm specific time-intensity data were used to determine Q̇DIA by two individuals. Transdiaphragmatic pressure for unloaded and each loading stage were 15.2 ± 0.8, 26.1 ± 0.8, 34.6 ± 0.8, and 40.0 ± 0.8 % of max, respectively. Q̇DIA increased with each stage of loading (3.1 ± 3.1, 6.9 ± 3.6, 11.0 ± 4.9, and 13.5 ± 5.4 AU·s-1; P<0.0001). The linear relationship between diaphragmatic flow and pressure was reproducible from day-to-day. Q̇DIA had good-to-excellent test-retest reliability (0.86 [0.77,0.92]; P<0.0001) and excellent inter-analyzer reproducibility (0.93 [0.90,0.95]; P<0.0001) with minimal bias. Relative Q̇DIA measurements have valid physiological underpinnings, are reliable day-to-day, and reproducible analyzer-to-analyzer. Contrast-enhanced ultrasound is a viable, minimally invasive method for assessing costal Q̇DIA in humans and may provide a tool to monitor diaphragm hemodynamics in clinical settings.