Near‐infrared diffuse correlation spectroscopy (DCS) is an emerging technique for non‐invasive measurement of local muscle blood flow at the microvascular level. We have previously shown excellent agreement between single wavelength DCS and Doppler ultrasound of the brachial artery during rhythmic handgrip exercise, supporting the role of DCS in exercise physiology. Here, we report novel DCS data from our lab, incorporating two‐different wavelengths (785 nm and 852 nm), allowing for direct assessment of microvascular perfusion, together with oxyhemoglobin and deoxyhemoglobin. To‐date, we have studied eight individuals (male/female: 3/5, mean: age 48±22 (range: 22–76 years), height 170±8 cm, and weight 75±12 kg). Subjects were instrumented with the DCS probe located over the belly of the flexor digitorum profundus. Duplex ultrasound of the brachial artery was also performed as a secondary measure of skeletal muscle blood flow. Each subject performed two bouts of rhythmic hand grip exercise at 20% of their maximum voluntary contraction (MVC). Each exercise bout was preceded by a standardized resting baseline, and each period of data collection was separated by at least 10 minutes of rest. Data from each round of data collection were averaged. As reported previously using our single wavelength DCS device, blood flow index (BFI, the primary output from DCS) increased significantly (119±37%) with exercise. We also observed a −1.9±1.1% change in oxyhemoglobin and 21.8±10.0% change in deoxyhemoglobin resulting in a −5.9±2.6% change in tissue saturation with exercise. As a result, relative muscle oxygen consumption (rmVO2) increased by 160.2±55.4%. The novelty of this new approach is best illustrated by a case‐comparison between two subjects, who performed similar absolute (11 vs 10 kg) and relative work (20%), and yet achieved disparate levels of oxygen utilization during exercise (ΔrmO2 = 307% vs. 214%, Case A vs. Case B respectively). This difference appears to be explained predominantly by muscle oxygen extraction as both brachial artery blood flow and microvascular perfusion (by DCS) were similar in both subjects. In contrast, Case A exhibited a much greater change in StO2 (−17.8%) compared to Case B, whose StO2 more closely mirrored the group average (−6.8%). To help interpret these results, we evaluated skeletal muscle oxidative capacity in both subjects using an established NIRS‐based cuff occlusion protocol (Rosenberry et al. 2018. JoVE). Remarkably, these additional data corroborated our assumptions, showing a muscle oxygen consumption recovery time of 34 seconds in Case A and 93 seconds in Case B. Taken together, these data establish strong proof‐of‐concept that dual wavelength DCS can provide important mechanistic insight into the determinants of oxygen consumption. Extending these findings to patients with exercise intolerance (e.g. heart failure with preserved or reduced ejection fraction) may provide important therapeutic insight.Support or Funding InformationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.