Skeletal muscle perfusion and oxygenation are commonly evaluated using Doppler ultrasound and near-infrared spectroscopy (NIRS) techniques. However, a recently developed magnetic resonance imaging (MRI) sequence, termed PIVOT, permits the simultaneous collection of skeletal muscle perfusion and T2* (an index of skeletal muscle oxygenation). Purpose To determine the level of agreement between PIVOT, Doppler ultrasound, and NIRS-based assessments of skeletal muscle perfusion and oxygenation. Methods Twelve healthy volunteers (8 females, 25 ± 3 years, 170 ± 11 cm, 71.5 ± 8.0 kg) performed six total reactive hyperemia protocols. During three of these reactive hyperemia protocols, Tissue Saturation Index (TSI) and oxygenated hemoglobin (O2Hb) were recorded from the medial gastrocnemius (MG) and tibialis anterior (TA), and blood flow velocity was recorded from the popliteal artery (BFvpop) via Doppler Ultrasound. The other three trials were performed inside the bore of a 3 T MRI scanner, and the PIVOT sequence was used to assess perfusion (PIVOTperf) and oxygenation (T2*) of the medial gastrocnemius and tibialis anterior muscles. Positive incremental areas under the curve (iAUC) and times to peak (TTP) were calculated for each variable, and the level of agreement between collection methods was evaluated via Bland-Altman analyses and Spearman’s Rho correlation analyses. Results The only significant bivariate relationships observed were between the T2* vs. TSI iAUC and PIVOTperf vs. BFvpop values recorded from the MG. Significant mean differences were observed for all comparisons (all P ≤ 0.038), and significant proportional biases were observed for the PIVOTperf vs. tHb TTP (R2 = 0.848, P textless 0.001) and T2* vs. TSI TTP comparisons in the TA (R2 = 0.488, P = 0.011), and the PIVOTperf vs. BFvpop iAUC (R2 = 0.477, P = 0.013) and time to peak (R2 = 0.851, P textless 0.001) comparisons in the MG. Conclusions Our findings suggest that the PIVOT technique has, at best, a moderate level of agreement with Doppler ultrasound and NIRS assessment methods and is subject to significant proportional bias. These findings do not challenge the accuracy of either measurement technique but instead reflect differences in the vascular compartments, sampling volumes, and parameters being evaluated.