In the present study, we examined how fatiguing exercise affects O2-based measures of skeletal muscle oxidative capacity in vivo by measuring changes in the rate constant of muscle V̇o2 recovery ([Formula: see text]). Healthy young adults completed isokinetic (120°·s-1), maximal voluntary dynamic contractions (MVDCs) lasting 24 (baseline [Formula: see text]) and 240 s (postfatiguing exercise [Formula: see text]). Vastus lateralis [Formula: see text] was measured using near-infrared diffuse correlation spectroscopy (NIRS-DCS) via the conventional repeated arterial occlusion method (part A, n = 14) or a novel NIRS-DCS "free-flow" method (part B, n = 13). Pulmonary V̇o2 (pV̇o2), muscle V̇o2 (mV̇o2), and surface electromyography (sEMG) measures of muscle activation were also measured throughout the 240-s trial. Compared with the 24-s trial, [Formula: see text] following 240 s of MVDCs was impaired by ∼25% (part A; P = 0.005) and ∼16% (part B; P = 0.017). Moreover, both pV̇o2 and mV̇o2 rapidly increased to maximal levels, where they remained for the duration of the 240-s trial, despite sEMG activity and peak MVDC power declining. These results demonstrate that fatiguing exercise not only impairs O2-based measures of skeletal muscle oxidative capacity, but also that mitochondrial O2 consumption is uncoupled from power output and ATP demand during fatiguing exercise.NEW & NOTEWORTHY We measured rates of skeletal muscle V̇o2 recovery ([Formula: see text]) at baseline and following fatiguing exercise using near-infrared diffuse correlation spectroscopy (NIRS-DCS). Regardless of whether [Formula: see text] was measured via the conventional repeated arterial occlusion method (part A) or a novel NIRS-DCS "free-flow" method (part B), fatiguing exercise impaired [Formula: see text] by ∼15%-25%. Because ATP demand rapidly declines post exercise, the slow [Formula: see text] recovery observed here suggests fatiguing exercise may uncouple the functional relationship between mitochondrial O2 consumption and ATP synthase activity.

Significance:Frequency-domain near-infrared spectroscopy (FD-NIRS) currently enables absolute hemoglobin quantification but requires multidistance measurements of both amplitude attenuation and phase shifts. Notably, existing FD-NIRS approaches have not demonstrated reliable quantification of differential redox-state concentrations of cytochrome c oxidase ( CCO redox ), a critical metabolic marker. Aim:We aimed to develop a novel optimization-based algorithm for single source-detector (S-D), phase-only FD-NIRS that achieves accurate quantifications of hemoglobin parameters (HbO and Hb) and CCO redox . Approach:Our computational framework implemented both forward modeling and inverse reconstruction. For the modeling, we first defined chromophore concentration sets (HbO, Hb, CCO redox ), followed by calculations of wavelength-dependent optical properties for two- or eight-wavelength configurations. Next, time-domain photon propagation was generated via Monte Carlo (MC) simulations, and FD-NIRS parameters (modulation amplitude, phase) were extracted through Fourier analysis. In the inverse computation, nonlinear optimization with edge-barrier regularization was employed for the recovery of chromophore concentrations. Both the multiseparation method and the single S-D, phase-only algorithm were used to reconstruct chromophore concentrations. Results:Respective performances evaluated for the two methods were compared through their concentration recovery accuracy. In either the two- or eight-wavelength configuration, our new algorithm outperformed the conventional method for the S-D separations up to 3 cm for all three chromophores. In particular, CCO redox estimation was improved markedly from a mean relative error of 34.1% with the conventional method to just 5.1% using our algorithm. Conclusions:These results validate single-separation phase-only FD-NIRS as an accurate method for multichromophore quantification (including CCO redox ), enabling simpler, cost-effective systems without compromising metabolic imaging capability. The approach achieves < 10 % error in hemoglobin quantification while eliminating traditional multidistance requirements.

BACKGROUND: We identified peripherally limited patients using cardiopulmonary exercise testing and measured skeletal muscle oxygen transport and utilization during invasive single leg exercise testing to identify the mechanisms of the peripheral limitation. METHODS: Forty-five patients with heart failure with preserved ejection fraction (70 +/- 7 years, 27 females) completed seated upright cardiopulmonary exercise testing and were defined as having a (1) peripheral limitation to exercise if cardiac output/oxygen consumption (VO2) was elevated (>= 6) or 5 to 6 with a stroke volume reserve >50% (n=31) or (2) a central limitation to exercise if cardiac output/VO2 slope was <= 5 or 5 to 6 with stroke volume reserve <50% (n=14). Single leg knee extension exercise was used to quantify peak leg blood flow (Doppler ultrasound), arterial-to-venous oxygen content difference (femoral venous catheter), leg VO2, and muscle oxygen diffusive conductance. In a subset of participants (n=36), phosphocreatine recovery time was measured by magnetic resonance spectroscopy to determine skeletal muscle oxidative capacity. RESULTS: Peak VO2 during cardiopulmonary exercise testing was not different between groups (central: 13.9 +/- 5.7 versus peripheral: 12.0 +/- 3.1 mL/min per kg; P=0.135); however, the peripheral group had a lower peak arterial-to-venous oxygen content difference (central: 13.5 +/- 2.0 versus peripheral: 11.1 +/- 1.6 mLO(2)/dL blood; P<0.001). During single leg knee extension, there was no difference in peak leg VO2 (P=0.306), but the peripherally limited group had greater blood flow/VO2 ratio (P=0.024), lower arterial-to-venous oxygen content difference (central: 12.3 +/- 2.5 versus peripheral: 10.3 +/- 2.2 mLO(2)/dL blood; P=0.013), and lower muscle oxygen diffusive conductance (P=0.021). A difference in magnetic resonance spectroscopy-derived phosphocreatine recovery time was not detected (P=0.199). CONCLUSIONS: Peripherally limited patients with heart failure with preserved ejection fraction identified by cardiopulmonary exercise testing have impairments in oxygen transport and utilization at the level of the skeletal muscle quantified by invasive knee extension exercise testing, which includes an increased blood flow/VO2 ratio and poor muscle diffusive capacity.

PURPOSE:Our aim was to design and build a 3T 31P/1H calf coil that is capable of providing both good 31P and 1H transmit and receive performance, as well as being capable of accommodating a near-infrared spectroscopy (NIRS) device for simultaneous NIRS data and MRI/MRS acquisition. METHOD:In this work, we propose a new 3T 31P/1H birdcage combination design consisting of two co-centrically positioned birdcages on the same surface to maximize transmit efficiency and sensitivity for both nuclei. The 31P birdcage is a high-pass birdcage, whereas the 1H birdcage is a low-pass one to minimize coupling. The diameter of the 31P/1H birdcage combination was designed to be large enough to accommodate a NIRS device for simultaneous NIRS data and MRI/MRS acquisition. RESULTS:The one-layer coil structure of the birdcage combination significantly streamlines the mechanical design and coil assembly process. Full-wave simulation results show that the 31P and 1H are very well decoupled with each other, and the 1H and 31P SNR surpasses that of their standalone counterparts in the central area. Experiment results show that the inclusion of a NIRS device does not significantly affect the performance of the coil, thus enabling simultaneous NIRS and MRI readouts during exercise. CONCLUSION:Our findings demonstrate the feasibility and effectiveness of this dual-tuned coil design for combined NIRS and MRS measurements, offering potential benefits for studying metabolic and functional changes in the skeletal muscle in vivo.

Significance:Diffuse correlation spectroscopy (DCS) permits non-invasive assessment of skeletal muscle blood flow but may misestimate changes in muscle perfusion. Aim:We aimed to highlight recent evidence that DCS blood flow index (BFI) misestimates changes in muscle blood flow during physiological perturbation and to introduce a novel approach that adjusts BFI for estimated changes in vasodilation. Approach:We measured changes in muscle BFI during quadriceps and forearm exercises using DCS, the latter of which were adjusted for estimated changes in microvascular flow area and then compared to Doppler ultrasound in the brachial artery. Then, we compared adjusted BFI- and arterial spin labeling (ASL) MRI measures of gastrocnemius blood flow during reactive hyperemia and plantar flexion exercise. Results:We observed little-to-no change in quadriceps BFI during maximal-effort exercise. Similarly, forearm BFI was modestly increased during handgrip exercise, but the magnitude was significantly lower than measured by Doppler ultrasound in the brachial artery. However, this difference was ameliorated after adjusting BFI for estimated changes in microvascular flow area. Similar observations were also observed in the gastrocnemius when directly comparing the adjusted BFI values to ASL-MRI. Conclusions:Adjusting BFI for estimated changes in microvascular flow area may improve DCS estimates of muscle blood flow, but further study is needed to validate these methods moving forward.

Near-infrared diffuse correlation spectroscopy (NIR-DCS) is an optical imaging technique for measuring relative changes in skeletal muscle microvascular perfusion (i.e., fold change above baseline) during reactive hyperemia testing and exercise and is reported as a blood flow index (BFI). Although it is generally accepted that changes in BFI are primarily driven by changes in muscle perfusion, it is well known that large, hyperthermia-induced changes in cutaneous blood flow can uncouple this relationship. What remains unknown, is how much of an impact that changes in cutaneous perfusion have on NIR-DCS BFI and estimates of skeletal muscle perfusion under thermoneutral conditions, where changes in cutaneous blood flow are assumed to be relatively low. We therefore used epinephrine iontophoresis to pharmacologically block changes in cutaneous perfusion throughout a battery of experimental procedures. The data show that 1) epinephrine iontophoresis attenuates changes in cutaneous perfusion for up to 4-h posttreatment, even in the face of significant neural and local stimuli, 2) under thermoneutral conditions, cutaneous perfusion does not significantly impact NIR-DCS BFI during reactive hyperemia testing or moderate-intensity exercise, and 3) during passive whole body heat stress, when cutaneous vasodilation is pronounced, epinephrine iontophoresis preserves NIR-DCS measures of skeletal muscle BFI during moderate-intensity exercise. Collectively, these data suggest that cutaneous perfusion is unlikely to have a major impact on NIR-DCS estimates of skeletal muscle BFI under thermoneutral conditions, but that epinephrine iontophoresis can be used to abolish cutaneous contamination of the NIR-DCS BFI signal during studies where skin blood flow may be elevated but skeletal muscle perfusion is of specific interest.

PURPOSE: Cardiac magnetic resonance imaging (cMRI) is ideal for non-invasive assessment of cardiovascular structure and function. Advancements in imaging technology and hardware development has made exercise cMRI more robust and increasingly more feasible. The purpose of this study was therefore three-fold: First, to compare the oxygen cost of upright cycling with supine exercise using a commercially available MRI compatible exercise ergometer. Second, to evaluate the bi-ventricular response to graded sub-maximal exercise using cMRI. Third, to evaluate the reproducibility of exercise cMRI across a broad range of exercise intensity. METHODS: To accomplish these goals, we first performed incremental exercise tests, on two separate days, using a conventional upright cycle ergometer and an MRI compatible stepping ergometer; beginning with three-minute stages (4 x 0.3 W/kg/stage), followed by one-minute stages to exhaustion (0.2 W/kg/stage). Then, on a separate visit, participants exercised inside the bore of a 3 T MRI at 20-, 40-, and 60% of their upright maximum. Left ventricular stroke volume was assessed by the method-of-disks, using high-resolution short-axis cine imaging, with brief (~2-3 sec) end-expiratory pauses. To assess the reproducibility of exercise cMRI, imaging was repeated on a separate visit. RESULTS: Peak oxygen consumption, peak heart rate, and peak work rate were lower during supine stepping exercise with the MRI-compatible ergometer than during upright cycle exercise (supine: upright = 86 ± 7%, 93 ± 8%, and 91 ± 9%). The metabolic cost of performing submaximal exercise, however, was very similar between the two ergometers (0.21 ± 0.06 ml/kg/min per watt versus 0.20 ± 0.05 ml/kg/min per watt, upright versus supine, respectively; p = 0.34). During exercise cMRI, left ventricular stroke volume increased from rest with 20% and 40% exercise, returning towards baseline at 60% (100.7 ± 21.5 mL, 114.6 ± 29.0 mL, 116.6 ± 33.8 mL, 104.2 ± 27.7 mL, rest, 20%, 40%, and 60%, respectively), with excellent agreement between trials (ICC =0.96). CONCLUSION: Taken together, these results support the use of exercise cMRI for evaluation of cardiovascular structure and function, establishing both feasibility and reproducibility.Funded by NIH (P01HL137630) and the Potratz Family Endowment.

Abstract We evaluated whether task‐dependent, age‐related differences in muscle fatigue (contraction‐induced decline in normalized power) develop from differences in bioenergetics or metabolic economy (ME; mass‐normalized work/mM ATP). We used magnetic resonance spectroscopy to quantify intracellular metabolites in vastus lateralis muscle of 10 young and 10 older adults during two maximal‐effort, 4‐min isotonic (20% maximal torque) and isokinetic (120°s−1) contraction protocols. Fatigue, inorganic phosphate (Pi), and pH (p ≥ 0.213) differed by age during isotonic contractions. However, older had less fatigue (p ≤ 0.011) and metabolic perturbation (lower [Pi], greater pH; p ≤ 0.031) than young during isokinetic contractions. ME was lower in older than young during isotonic contractions (p ≤ 0.003), but not associated with fatigue in either protocol or group. Rather, fatigue during both tasks was linearly related to changes in [H+], in both groups. The slope of fatigue versus [H+] was 50% lower in older than young during isokinetic contractions (p ≤ 0.023), consistent with less fatigue in older during this protocol. Overall, regardless of age or task type, acidosis, but not ME, was the primary mechanism for fatigue in vivo. The source of the age‐related differences in contraction‐induced acidosis in vivo remains to be determined, as does the apparent task‐dependent difference in the sensitivity of muscle to [H+].

PURPOSE Exercise intolerance is the hallmark symptom of heart failure with preserved ejection fraction (HFpEF), the most common form of heart failure among older individuals. Initially considered to be a central limitation (i.e. abnormal left ventricular relaxation and impaired cardiac output), emerging evidence supports a peripheral limitation to exercise. However, direct evaluation of the quadriceps (the primary locomotor muscle group) remains limited. METHODS HFpEF patients (n = 23, 12F, 68.1 ± 6.4 yrs) and age-matched controls (n = 23, 14F, 69.2 ± 6.6 yrs) completed: 1) an incremental cycle test to assess VO2max, 2) T2- magnetic resonance imaging to quantify thigh muscle volume and fat content, and 3) 31P magnetic resonance spectroscopy of the quadriceps to quantify muscle oxidative capacity; measured as the Tau (τ) of phosphocreatine recovery (τPCr) immediately following knee extension exercise. Whole-body arterial-venous oxygen difference and cardiac output were also measured in HFpEF patients (but not controls) to further sub-divide HFpEF patients as being centrally- (Type A) versus peripherally-limited (Type-B). RESULTS HFpEF had greater body mass (p = 0.001) and BMI (p = 0.001) than Controls, and a lower VO2max when normalized to body mass (14.3 ± 4.5 vs 18.9 ± 4.6 ml/kg/min; p = 0.006), but no differences were observed for VO2max normalized to leg lean mass (899.5 ± 168.3 vs 982.8 ± 164.1 ml/kg/min; p = 0.096), τPCr (57.1 ± 11.8 vs 54.4 ± 14.9 s; p = 0.518), or muscle- (22.5 ± 7.0 vs 19.9 ± 4.0 %; p = 0.127) and thigh-fat percentages (56.1 ± 16.0 vs 51.4 ± 12.4 %; p = 0.287). After separating Type-A (n = 8, 1F) and Type-B (n = 15, 11F) HFpEF patients, VO2max normalized to leg mass (main effect p = 0.115) and τPCr (main effect p = 0.481) were still not different between groups, but Type-B HFpEF patients did exhibit greater muscle- (24.9 ± 7.0 %) and thigh-fat (62.3 ± 13.7 %) percentages than Type-A HFpEF patients (18.1 ± 4.6 and 44.5 ± 14.0 %; p < 0.015) and Controls (p < 0.05). CONCLUSION The results of the present study do not support impairments in intrinsic skeletal muscle oxidative capacity as a major source of exercise intolerance in HFpEF but highlight potentially important roles for differences in skeletal muscle- and thigh-fat content. This study was funded by the National Institutes of Health (P01 HL137630).

Human skeletal muscle oxidative capacity can be quantified non-invasively using 31-phosphorus magnetic resonance spectroscopy (31P-MRS) to measure the rate constant of phosphocreatine (PCr) recovery ( kPCr) following contractions. In the quadricep muscles, several studies have quantified kPCr following 24–30 s of sustained maximal voluntary isometric contraction (MVIC). This approach has the advantage of simplicity but is potentially problematic because sustained MVICs inhibit perfusion, which may limit muscle oxygen availability or increase the intracellular metabolic perturbation, and thus affect kPCr. Alternatively, dynamic contractions allow reperfusion between contractions, which may avoid limitations in oxygen delivery. To determine whether dynamic contraction protocols elicit greater kPCr than sustained MVIC protocols, we used a cross-sectional design to compare quadriceps kPCr in 22 young and 11 older healthy adults following 24 s of maximal voluntary: (1) sustained MVIC and (2) dynamic (MVDC; 120°·s−1, 1 every 2 s) contractions. Muscle kPCr was ∼20% lower following the MVIC protocol compared with the MVDC protocol ( p ≤ 0.001), though this was less evident in older adults ( p = 0.073). Changes in skeletal muscle pH ( p ≤ 0.001) and PME accumulation ( p ≤ 0.001) were greater following the sustained MVIC protocol, and pH ( p ≤ 0.001) and PME ( p ≤ 0.001) recovery were slower. These results demonstrate that (i) a brief, sustained MVIC yields a lower value for skeletal muscle oxidative capacity than an MVDC protocol of similar duration and (ii) this difference may not be consistent across populations (e.g., young vs. old). Thus, the potential effect of contraction protocol on comparisons of kPCr in different study groups requires careful consideration in the future.

Key points The oxygen cost of high-intensity exercise at power outputs above an individual's lactate threshold (LT) is greater than would be predicted by the linear oxygen consumption-power relationship observed below the LT. However, whether these augmentations are caused by an increased ATP cost of force generation (ATP(COST)) or an increased oxygen cost of ATP synthesis is unclear. We used P-31-MRS to measure changes in cytosolic [ADP] (intramyocellular marker of oxidative metabolism), oxidative ATP synthesis (ATP(OX)) and ATP(COST) during a 6-stage, stepwise knee extension protocol. ATP(COST) was unchanged across stages. The relationship between [ADP] and muscle power output was augmented at workloads above the pH threshold (pH(T); proxy for LT), whereas increases in ATP(OX) were attenuated. These results suggest the greater oxygen cost of contractions at workloads beyond the pH(T) is not caused by mechanisms that increase ATP(COST), but rather mechanisms that alter intrinsic mitochondrial function or capacity. Increases in skeletal muscle metabolism and oxygen consumption are linearly related to muscle power output for workloads below the lactate threshold (LT), but are augmented (i.e. greater rate of increase relative to workload) thereafter. Presently, it is unclear whether these metabolic augmentations are caused by increases in the ATP cost of force generation (ATP(COST)) or changes in the efficiency of mitochondrial oxygen consumption and oxidative ATP synthesis (ATP(OX)). To partition these two hypotheses in vivo, we used P-31-MRS to calculate slopes relating step-changes in muscle work to concurrent changes in cytosolic phosphates and ATP(OX) before and after the pH threshold (pH(T); used here as a proxy for LT) within the vastus lateralis muscle of eight young adults during a stepwise knee extension test. Changes in muscle phosphates and ATP(OX) were linearly related to workload below the pH(T). However, slopes above the pH(T) were greater for muscle phosphates (P < 0.05) and lower for ATP(OX) (P < 0.05) than were the slopes observed below the pH(T). The maximal capacity for ATP(OX) (V?max) and ADP-specific ATP(OX) also declined beyond the pH(T) (P < 0.05), whereas ATP(COST) was unchanged (P = 0.10). These results oppose the hypothesis that high-intensity contractions increase ATP(COST) and suggest that greater oxidative metabolism at workloads beyond the pH(T) is caused by mechanisms that affect intrinsic mitochondrial function or capacity, such as alterations in substrate selection or electron entry into the electron transport chain, temperature-mediated changes in mitochondrial permeability to protons, or stimulation of mitochondrial uncoupling by reactive oxygen species generation.

We used passive whole body heat stress, in combination with local intradermal botulinum toxin type A treatment, to experimentally manipulate cutaneous blood flow and investigate its impact on NIR-DCS measures of skeletal muscle BFI at rest and during exercise. Collectively, the results show that cutaneous blood flow, which was augmented in response to passive whole body heat stress, markedly affects NIR-DCS-derived BFI, such that the BFI signal becomes dominated by changes in cutaneous red blood cell flux.

Near-infrared diffuse correlation spectroscopy (DCS) is increasingly used to study relative changes in skeletal muscle blood flow. However, most diffuse correlation spectrometers assume that tissue optical properties-such as absorption (μa) and reduced scattering (μ's) coefficients-remain constant during physiological provocations, which is untrue for skeletal muscle. Here, we interrogate how changes in tissue μa and μ's affect DCS calculations of blood flow index (BFI). We recalculated BFI using raw autocorrelation curves and μa/μ's values recorded during a reactive hyperemia protocol in 16 healthy young individuals. First, we show that incorrectly assuming baseline μa and μ's substantially affects peak BFI and BFI slope when expressed in absolute terms (cm2/s, P < 0.01), but these differences are abolished when expressed in relative terms (% baseline). Next, to evaluate the impact of physiologic changes in μa and μ's, we compared peak BFI and BFI slope when μa and μ's were held constant throughout the reactive hyperemia protocol versus integrated from a 3-s rolling average. Regardless of approach, group means for peak BFI and BFI slope did not differ. Group means for peak BFI and BFI slope were also similar following ad absurdum analyses, where we simulated supraphysiologic changes in μa/μ's. In both cases, however, we identified individual cases where peak BFI and BFI slope were indeed affected, with this result being driven by relative changes in μa over μ's. Overall, these results provide support for past reports in which μa/μ's were held constant but also advocate for real-time incorporation of μa and μ's moving forward.NEW & NOTEWORTHY We investigated how changes in tissue optical properties affect near-infrared diffuse correlation spectroscopy (NIR-DCS)-derived indices of skeletal muscle blood flow (BFI) during physiological provocation. Although accounting for changes in tissue optical properties has little impact on BFI on a group level, individual BFI calculations are indeed impacted by changes in tissue optical properties. NIR-DCS calculations of BFI should therefore account for real-time, physiologically induced changes in tissue optical properties whenever possible.

Key points We used 31‐phosphorus magnetic resonance spectroscopy to quantify in vivo skeletal muscle metabolic economy (ME; mass‐normalized torque or power produced per ATP consumed) during three 24 s maximal‐effort contraction protocols: (1) sustained isometric (MVIC), (2) intermittent isokinetic (MVDCIsoK), and (3) intermittent isotonic (MVDCIsoT) in the knee extensor muscles of young and older adults. ME was not different between groups during the MVIC but was lower in older than young adults during both dynamic contraction protocols. These results are consistent with an increased energy cost of locomotion, but not postural support, with age. The effects of old age on ME were not due to age‐related changes in muscle oxidative capacity or ATP flux. Specific power was lower in older than young adults, despite similar total ATP synthesis between groups. Together, this suggests a dissociation between cross‐bridge activity and ATP utilization with age. AbstractMuscle metabolic economy (ME; mass‐normalized torque or power produced per ATP consumed) is similar in young and older adults during some isometric contractions, but less is known about potential age‐related differences in ME during dynamic contractions. We hypothesized that age‐related differences in ME would exist only during dynamic contractions, due to the increased energetic demand of dynamic versus isometric contractions. Ten young (Y; 27.5 ± 3.9 years, 6 men) and 10 older (O; 71 ± 5 years, 5 men) healthy adults performed three 24 s bouts of maximal contractions: (1) sustained isometric (MVIC), (2) isokinetic (120°·s–1, MVDCIsoK; 0.5 Hz), and (3) isotonic (load = 20% MVIC, MVDCIsoT; 0.5 Hz). Phosphorus magnetic resonance spectroscopy of the vastus lateralis muscle was used to calculate ATP flux (mM ATP·s–1) through the creatine kinase reaction, glycolysis and oxidative phosphorylation. Quadriceps contractile volume (cm3) was measured by MRI. ME was calculated using the torque‐time integral (MVIC) or power‐time integral (MVDCIsoK and MVDCIsoT), total ATP synthesis and contractile volume. As hypothesized, ME was not different between Y and O during the MVIC (0.12 ± 0.03 vs. 0.12 ± 0.02 Nm.s.cm–3.mM ATP–1, mean ± SD, respectively; P = 0.847). However, during both MVDCIsoK and MVDCIsoT, ME was lower in O than Y adults (MVDCIsoK: 0.011 ± 0.003 vs. 0.007 ± 0.002 J.cm–3.mM ATP–1; P < 0.001; MVDCIsoT: 0.011 ± 0.002 vs. 0.008 ± 0.002; P = 0.037, respectively), despite similar muscle oxidative capacity, oxidative and total ATP flux in both groups. The lower specific power in older than young adults, despite similar total ATP synthesis between groups, suggests there is a dissociation between cross‐bridge activity and ATP utilization with age.

Postischemia reperfusion kinetics are markedly dissociated when comparing the macro- versus microvasculature. We used Doppler ultrasound and near-infrared diffuse correlation spectroscopy (NIR-DCS), an emerging technique for continuously and noninvasively quantifying relative changes in skeletal muscle microvascular perfusion (i.e., blood flow index or BFI), to measure macro- and microvascular reactive hyperemia (RH) in the nondominant arm of 16 healthy young adults. First, we manipulated the duration of limb ischemia (3 vs. 6 min) with the limb at heart level (neutral, -N). Then, we reduced/increased forearm perfusion pressure (PP) by positioning the arm above (3 min-A, 60°) or below (3 min-B, 30°) the heart. The major novel findings were twofold: first, changes in the ischemic stimulus similarly affected peak macrovascular (i.e., conduit, mL/min) and microvascular (i.e., peak NIR-DCS-derived BFI) reperfusion during reactive hyperemia (6 min-N > 3 min-N, P < 0.05, both) but did not affect the rate at which microvascular reperfusion occurs (i.e., BFI slope). Second, changing forearm PP predictably affected both peak macro- and microvascular reperfusion during RH (3 min-B > N > A, P < 0.05, all), as well as the rate at which microvascular reperfusion occurred (BFI slope; 3 min-B >N > A, P < 0.05). Together, the data suggest that kinetic differences between macro- and microvascular reperfusion are largely determined by differences in fluid mechanical energy (i.e., pressure, gravitational, and kinetic energies) between the two compartments that work in tandem to restore pressure across the arterial tree following a period of tissue ischemia.NEW & NOTEWORTHY We extend our understanding of macro- versus microvascular hemodynamics in humans, by using near-infrared diffuse correlation spectroscopy (micro-) and Doppler ultrasound (macro-) to characterize reperfusion hemodynamics following experimental manipulation of the ischemic stimulus and tissue perfusion pressure. Our results suggest kinetic differences between macro- and microvascular reperfusion are largely determined by differences in fluid mechanical energy (i.e., pressure, gravitational, and kinetic energies) between the two compartments, rather than inherent differences between the macro- and microvasculature.

Although high-velocity contractions elicit greater muscle fatigue in older than young adults, the cause of this difference is unclear. We examined the potential roles of resting muscle architecture and baseline contractile properties, as well as changes in voluntary activation and low-frequency fatigue in response to high-velocity knee extensor work. Vastus lateralis muscle architecture was determined in quiescent muscle by ultrasonography in 8 young (23.4±1.8 yrs) and 8 older women (69.6±1.1). Maximal voluntary dynamic (MVDC) and isometric (MVIC), and stimulated (80Hz and 10Hz, 500ms) isometric contractions were performed before and immediately after 120 MVDCs (240°.s-1, one every 2s). Architecture variables did not differ between groups (p≥0.209), but the half-time of torque relaxation (T1/2) was longer in older than young women at baseline (151.9±6.0 vs. 118.8±4.4 ms, respectively, p = 0.001). Older women fatigued more than young (to 33.6±4.7% vs. 55.2±4.2% initial torque, respectively; p = 0.004), with no evidence of voluntary activation failure (ΔMVIC:80Hz torque) in either group (p≥0.317). Low-frequency fatigue (Δ10:80Hz torque) occurred in both groups (p<0.001), as did slowing of T1/2 (p = 0.001), with no differences between groups. Baseline T1/2 was inversely associated with fatigue in older (r2 = 0.584, p = 0.045), but not young women (r2 = 0.147, p = 0.348). These results indicate that differences in muscle architecture, voluntary activation, and low-frequency fatigue do not explain the greater fatigue of older compared with young women during high-velocity contractions. The inverse association between baseline T1/2 and fatigue in older women suggests that factors related to slower muscle contractile properties may be protective against fatigue during fast, repetitive contractions in aging.

A magnetic resonance (MR) compatible ergometer has been developed to study contracting lower limb muscles during acquisition of MR spectroscopy data, a technique to noninvasively measure metabolic energy in muscle tissue. Current active and passive MR-compatible ergometer designs lack torque or velocity control to allow precise mechanical measurements during isotonic and isokinetic contractions; incorporating load and velocity controllers while maintaining MR-compatibility is the main challenge. Presented in this paper is the design and evaluation of an MR-compatible ergometer designed to control knee torque or velocity up to 420 N.m and 270deg/s and is able to operate in a 3 Tesla magnetic field. The ergometer comprising of a passive component with no electronics or ferrous materials is located inside the bore of the scanner. The active component with the electronics and actuator located outside of the magnetic field in an adjacent room. The active components connect to the passive components via a cable that passes through the waveguide, a hole in the wall of the scanner room. System evaluations were performed and human subject evaluations were performed that measured the mechanical performance and show the mean percent errors below 9% in isotonic and 2% in isokinetic conditions.

Importantly, FMD is a prognostic indicator of future cardiovascular disease (Areas et al. 2019) and is quantified clinically during ischaemia–reperfusion testing. The magnitude of FMD (i.e. percentage change in arterial diameter) is often normalized to to account for inter-individual differences in the magnitude of post-ischaemic reperfusion (i.e. shear stress stimulus), under the assumption that blood viscosity does not change. However, blood is a non-Newtonian fluid, meaning viscosity will vary in response to changes in . Leo et al. recently demonstrated this concept by showing that blood viscosity decreased in response to increased during forearm exercise (Leo et al. 2020). Consequently, these findings have significant implications for clinical tests of FMD, because if changes in blood viscosity affect shear stress during ischaemia–reperfusion testing, then may be an inappropriate surrogate for normalizing FMD responses to τ. To address this gap in the literature, in a recent issue of The Journal of Physiology, Hoiland et al. examined post-ischaemic FMD and reactive hyperaemia in the brachial artery of 12 healthy young men before and after isovolumic haemodilution (Hoiland et al. 2020). Briefly, the protocol involved two ischaemia–reperfusion trials consisting of 1 min baseline, 5 min ischaemia and 3 min of recovery. The cuff was positioned just distal to the elbow joint and Doppler ultrasound was used to measure changes in brachial artery blood velocity and diameter. Following the first ischaemia–reperfusion trial, subjects then underwent an isovolumic haemodilution protocol in which ∼20% of the subject's whole blood was replaced with a 5% human serum albumin solution. The success of the haemodilution protocol was confirmed via repeated testing of haematocrit. Moreover, echocardiograms and blood draws were performed before each ischaemia–reperfusion test to examine changes in cardiac function and blood haemoglobin and nitric oxide levels, respectively. Although the isovolumic haemodilution protocol significantly reduced blood viscosity by ∼20%, shear stress during the post-ischaemic reperfusion period was not different between the control and haemodilution trials. However, shear rate was greater during the haemodilution trial due to greater rates of brachial artery blood flow. Interestingly, despite the similar shear stress stimuli, FMD was augmented following haemodilution compared to the control trial (+9.7% vs. +3.8%). Given that haemodilution reduced blood viscosity, which should reduce shear stress (eqn (1)) and FMD, the authors concluded that the augmented FMD response following haemodilution was due to the reduced scavenging of nitric oxide by haemoglobin. These data are further supported by a marked reduction in nitric oxide metabolites following haemodilution. Despite these compelling results, additional figures illustrating how brachial artery diameter, blood velocity and blood flow changed over time during reperfusion would have aided the interpretation of data, especially given that the temporal changes in shear stress were similar between the control and haemodilution trials, but that peak FMD was achieved ∼30 s later. Perhaps shear rate, on its own, is more important to stimulating FMD than shear stress, which would suggest that the augmented FMD observed during the haemodilution trial was a function of prolonged/augmented shear rates. Nevertheless, the strengths of this study far outweighed the minor consideration described above. The isovolumic replacement of whole blood with human serum albumin is indeed a unique and innovative strategy to experimentally manipulate blood viscosity without affecting cardiac output or blood pressure (as evidenced by the data presented). Moreover, differences in FMD were clearly not a function of the long supine rest period between haemo-conditions, as FMD did not change in a cohort of 11 young men who underwent the same protocol without isovolumic haemodilution (i.e. time control experiment). That Hct, [Hb], viscosity and NO levels were measured before each trial, allowed explicit demonstration of isovolumic haemodilution. The results and experimental approach described by Hoiland et al. (2020) stimulate several new and exciting lines of research. Indeed, the most interesting observation in the present study is the apparent increase in FMD following isovolumic haemodilution, despite near identical estimates of shear stress throughout the reperfusion period between the control and haemodilution trials. These similar shear stress profiles persisted regardless of isovolumic haemodilution reducing blood viscosity, which should reduce shear stress (eqn (1)). While reduced NO scavenging by haemoglobin is a very plausible explanation, as put forth by the investigators, it is interesting to consider the role of reduced arterial oxygen content (and oxygen delivery) on the present results. Indeed, our laboratory has recently highlighted the importance of the ‘ischaemic stimulus’ on reactive hyperaemia (Rosenberry et al. 2019). While the between condition difference in peak reactive hyperaemia did not reach statistical significance, there was a clear trend favouring the haemodilution condition. Whether this was the result of a greater ischaemic stimulus to vasodilate, and the extent to which it influenced the present results, should be considered in future experiments. It is also interesting to consider the role of resistive forces on fluid dynamics. Future studies will indeed need to delineate whether isovolumic haemodilution augments FMD purely as a function of reduced NO stimulation, or if reductions in the blood internal flow resistance also stimulate greater FMD through greater shear rates. To deductively separate the two, new experimental designs will be necessary. For instance, repeating the isovolumic condition with varying degrees of nitric oxide synthase inhibition and/or physical manipulation of shear rate (e.g. limb position changes, arterial compression). The results of this study also have important clinical implications. For example, brachial artery FMD is an indicator of future cardiovascular disease due to its correlation with coronary artery FMD (Takase et al. 1998). Given that Hoiland et al. demonstrated lower haematocrit and haemoglobin concentrations were associated with greater FMD, we would expect these factors should also improve coronary endothelial function. However, reduced haemoglobin is associated with increased risk of hospitalization and all-cause mortality in chronic heart failure patients (Tang & Katz, 2006). More work is therefore needed before we can fully integrate the present results into the clinical setting. Perhaps the biggest question raised from this important work is: is it necessary to measure haemoglobin concentration when performing routine ischaemia–reperfusion testing? At present, this is not typically performed. Hoiland et al. sought to assess the influence of haematocrit/haemoglobin on FMD in healthy humans via isovolumic haemodilution. Their results suggest that haemoglobin is an important factor in regulating FMD by constraining endothelium-dependent NO-mediated vasodilatation. The data also raise several new important questions, stimulating an existing area of future research. No competing interests declared. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. None.

Recent work has shown that measures of reactive hyperemia (RH) are temporally dissociated when assessed by Doppler ultrasound (i.e. upstream conduit blood flow) and near‐infrared diffuse correlation spectroscopy (NIR‐DCS; a direct measure of microvascular perfusion). To probe the physiological mechanisms underlying these discordant responses, we used Doppler ultrasound (Vivid‐i, GE) and NIR‐DCS (MetaOx, ISS Inc.) to measure RH in 13 healthy young adults (7F, 22.9 ± 3.3 yrs) under varying physiological conditions. First, with the arm at heart level (N), we manipulated the duration of limb ischemia (3 vs 6 min) to assess the impact of the ischemic stimulus. Second, we reduced/increased forearm perfusion pressure (FPP) by positioning the experimental arm above (60°) or below (30°) the heart, and measured RH following 3 min of occlusion. The 6min‐N trial was always conducted first, followed by the three 3‐min trials in randomized order. FPP was measured using finger photoplethysmography, corrected for the hydrostatic distance. For the 6min‐ and 3min‐N trials, resting BA blood flow was not different between conditions; however, baseline NIR‐DCS blood flow index (BFI) was lower during the 6min‐N trial than the 3min‐N trial (0.70 ± 0.40 vs 0.94 ± 0.41 cm2·10−8 AU, p < 0.01). As expected, the magnitude of peak BA blood flow during RH increased with increasing ischemia duration (341.9 ± 94.5 vs 284.9 ± 80.1 mL/min, 6min‐ vs 3min‐N, p = 0.03), as was peak NIR‐DCS derived BFI (3.11 ± 1.3 vs 2.54 ± 0.75 cm2·10−8 AU, p = 0.03). For the arm Above/Below trials, FPP was significantly lower in the Above position compared to Below (76.0 ± 5.4 vs 107.9 ± 5.9 mmHg, p < 0.01). Prior to cuff inflation, raising the arm above the heart also reduced BFI relative to the Below position (0.70 ± 0.26 vs 0.99 ± 0.51 cm2·10−8 AU, p = 0.03), whereas BA blood flow was similar between arm positions (23.4 ± 13.8 vs 32.1 ± 11.2 mL/min, p = 0.12). Following cuff deflation, peak BA blood flow (379.5 ± 143.5 vs 227.0 ± 63.9 mL/min, p < 0.01) and peak BFI (2.98 ± 1.00 vs 1.76 ± 0.59 cm2·10−8 AU, p <0.01) were higher with the arm Below the level of the heart compared to Above. The difference in peak BA blood flow was abolished however, when expressed as a % change from baseline (1198.8 ± 320.7 vs 1260.2 ± 817.8 %, Below vs Above, p = 0.79), whereas the difference in peak BFI was not (360.8 ± 170 vs 259.4 ± 64.8 %, p = 0.03). Remarkably, the RH BFI slope was nearly 3‐fold higher in the Below position compared to Above (0.14 ± 0.08 vs 0.05 ± 0.02, p < 0.01), but much smaller when manipulating the duration of ischemia (0.12 ± 0.04 vs 0.09 ± 0.04 cm2·10−8·s−1 AU, 3min‐ vs 6min‐N, p = 0.02), though this latter observation may have been due to an order effect. In toto, these results support previous findings that RH is dependent on the ischemic stimulus, regardless of the assessment technique, as the peak rates of BA blood flow and BFI were greater in the 6min‐N trial compared to the 3min‐N trial. Alternatively, the rate of microvascular reperfusion (i.e. BFI slope) is less affected by the ischemic stimulus to vasodilate and is instead primarily affected by changes in perfusion pressure, suggesting this measure may offer greater insight into the physiological mechanisms underlying microvascular function and reactive hyperemia.