The assessment of skeletal muscle oxygenation and perfusion are common elements in many human physiology studies. Among other techniques, laboratories often employ Doppler ultrasound assessments of limb blood flow [[1], [2], [3], [4], [5]] and near-infrared spectroscopy (NIRS; [[6], [7], [8], [9]]) as methods for assessing tissue perfusion and oxygenation, respectively. The use of such noninvasive techniques to assess skeletal muscle perfusion and oxygen consumption has grown substantially as direct assessments of these parameters are invasive [[10], [11], [12], [13]]. While these methods have broad applications to human research, they are not without limitations. The collection of limb blood flow or flow velocity via Doppler ultrasound is generally limited to the assessment of large (i.e., femoral and brachial) conduit arteries and is unable to assess microvascular perfusion within the skeletal muscle directly. Likewise, the assessment of skeletal muscle oxygenation via NIRS is limited to a small, superficial sampling volume and can be influenced by the depth of the subcutaneous adipose layer [14]. Moreover, these methods cannot be used to target the same area of tissue with a high level of precision. Therefore, there exists a need for a comprehensive method of simultaneously collecting skeletal muscle perfusion and oxygenation across an entire cross-section of skeletal muscle.

One such method is the use of multiparametric magnetic resonance imaging (MRI). A particular approach, termed PIVOT (Perfusion, Intravascular Venous Oxygen saturation, and T2*), was previously developed for the evaluation of changes in perfusion and oxygenation levels within skeletal muscle vasculature. The PIVOT sequence [15] utilized a novel interleaving technique to simultaneously collect skeletal muscle perfusion (via arterial spin labeling (ASL)), T2* relaxation time (via multi-echo-gradient-recalled-echo [mGRE]), and intravenous oxygen saturation (via mGRE) with a temporal resolution of 2 s. The base design of PIVOT was a flow-alternating inversion recovery ASL perfusion sequence, with saturation pulses applied at the end of each TR to force the magnetization into a saturation-recovery mode (termed Saturation Inversion Recovery or SATIR) [16]. A mGRE acquisition was interleaved into the ASL postlabeling delay, allowing for the simultaneous collection of venous oxygen saturation and T2*. The location of the mGRE slice was placed distal (~30 mm) to the ASL slice to avoid contamination of the perfusion measurement [15].

The techniques interleaved within a PIVOT acquisition have been broadly applied to study skeletal.

muscle physiology, either through the use of PIVOT or as a single parameter measurement (i.e., ASL or mGRE alone). ASL has been extensively used to collect in-vivo perfusion measurements in human skeletal muscle [[17], [18], [19], [20]] and cerebral tissue [[21], [22], [23]]. The acquisition of T2* and mixed venous oxygen saturation is especially important for the quantification of skeletal muscle microvascular function, as the paramagnetic properties of deoxygenated hemoglobin (and to a lesser extent, the diamagnetic properties of oxygenated hemoglobin) significantly influence the T2* signal intensity in a concentration-dependent manner [[24], [25], [26], [27]]. In light of this, T2* is regularly employed as an index of skeletal muscle oxygenation in studies involving both human [[28], [29], [30]] and animal models [31], where alterations in the ratio between deoxygenated and oxygenated hemoglobin occur. PIVOT itself was applied in several research studies as an alternative to Doppler ultrasound imaging and NIRS assessments of skeletal muscle perfusion and oxygenation [32,33].

As PIVOT continues to be employed in human physiology research, it is important to determine how well this imaging method agrees with more traditional laboratory-based methods. To that end, this study aimed to determine the level of agreement between Doppler ultrasound, NIRS, and PIVOT-based assessments of skeletal muscle perfusion and oxygenation, respectively. Based on differences in the vascular space analyzed during Doppler ultrasound and PIVOT assessments (i.e., conduit arteries vs. microvasculature), we expected the overall skeletal muscle perfusion responses and response times to differ significantly between these two methods. In contrast, we expected that NIRS and PIVOT-based assessments of skeletal muscle oxygenation would demonstrate a high level of agreement. This hypothesis is based on the presumption that a superficial portion of skeletal muscle would serve as a strong surrogate for oxygenation changes across the entire muscle cross-sectional area.