## 2 Materials and Methods
The diameter of the vessel was first measured with brightness (B)-mode via a screen shot image and the linear distance function. Next, blood flow velocity was determined via Doppler mode and a multi-frequency linear array probe (12L-Rs; 5-13 MHz; 38.4 mm field-of-view). Velocity was assessed at a constant insonation angle of 60\({}^{\circ}\) and the Doppler gate was set to the width of the vessel to promote complete insonation. Furthermore, velocity was recorded as the maximum frequency (TAMAX), or peak velocity, averaged across three complete cardiac cycles. Subsequently, this value was divided by two to produce a mean velocity [36; 37]. The mean velocity value was then multiplied by the cross-sectional area of the vessel to yield a measure of BABF [38]. Specifically, the equation used to determine BABF was as follows:  
\[BABF=\left(\frac{blood\ velocity\ (\mathrm{cm/s})}{2}\right)\times\pi\left( \frac{diameter\ (\mathrm{cm})}{2}\right)^{2}\times 60 \tag{1}\]  
A single researcher was responsible for collecting data with the ultrasound, and great care was given to avoid excess pressure on the vessel. A generous amount of water-soluble transmission gel was also used to enhance acoustic coupling and reduce artifact. Near-Infrared Spectroscopy. Measures derived from NIRS are based on tissue transparency for light in the near-infrared region (-700-900 nm), which allows devices to record the relative concentrations and oxygenation status of light-absorbing chromophores (e.g., hemoglobin, myoglobin) in skeletal muscle tissue [23]. The modified Beer-Lambert Law, as follows:  
\[A(OD)=Log(I_{0}/I)=\varepsilon\ [c]\ L\cdot DPF+G \tag{2}\]  
was used to account for light scattering [23], and, here, we used a differential pathlength factor (DPF) of 4 (i.e., the extended path length due to scattering) and a \(G\) constant (i.e., the loss of light due to scattering) [23; 32]. In the present study, the NIRS parameters (e.g., O\({}_{2}\)Hb, HHb, etc.) were collected from the T \(\times\) 2 channel at a sampling rate of 10 Hz, and the measures were tracked as changes from baseline [23; 32]. It is relevant to note the used channel given that this dictates signal depth. All values were relative to the initial baseline, and not absolute values. These values were used to calculate tHb (O\({}_{2}\)Hb + HHb) and HbDiff (O\({}_{2}\)Hb-HHb). To support the appropriateness of the NIRS forearm placement site, the ultrasound machine was used to quantify ATT. It was determined that each subject exhibited less than 2 cm of subcutaneous fat (mean \(\pm\) SD: 0.26 \(\pm\) 0.10 cm), rending T \(\times\) 2 an appropriate channel. The NIRS device was attached to the dominant forearm flexor digitorum superficialis muscle with black self-adhesive CobanTM in order to minimize exogenous light from influencing the signal. The specific device used was a portable,dual-wavelength (760 nm and 850 nm) continuous-wave, spatially resolved NIRS (Portalite, Artinis Medical Systems, Elst, The Netherlands). Throughout data collection, signal quality was assessed with continuous inspection of the signal fit factor and the percentage of light reaching the photodiode. Venous Occlusion Plethysmography. This procedure involved the application of a mercury in-silastic strain-gauge wrapped around the widest portion of the participant's dominant forearm, adjacent to the NIRS device. Great care was taken to minimize potential sources of error, such that the same researcher was responsible for the placement of the NIRS device and mercury in-silastic strain-gauge. The mercury in-silastic strain-gauge was selected based on measured forearm circumference. Prior to attaching the strain-gauge, the equipment was calibrated. A 2-point calibration was used to zero the baseline and set the high voltage point to 60. Afterwards, the units reflected mL of blood per 100 mL of tissue per minute (mL-dL \(\mathrm{FAV^{-1}\cdot min^{-1}}\)) [9; 39]. Thus, the strain-gauge provided a measure of blood flow via changes in circumference (i.e., strain-gauge length).