DCS blood flow index underestimates skeletal muscle perfusion in vivo: rationale and early evidence for the NIRS-DCS perfusion index.

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.

3T 31P/1H calf muscle coil for 1H and 31P MRI/MRS integrated with NIRS data acquisition.

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.

Measurements of in vivo skeletal muscle oxidative capacity are lower following sustained isometric compared with dynamic contractions.

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.

Muscle fatigue, bioenergetic responses and metabolic economy during load- and velocity-based maximal dynamic contractions in young and older adults.

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+ ].

Epinephrine iontophoresis attenuates changes in skin blood flow and abolishes cutaneous contamination of near-infrared diffuse correlation spectroscopy estimations of muscle perfusion.

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.

Impact of cutaneous blood flow on NIR-DCS measures of skeletal muscle blood flow index.

Near-infrared diffuse correlation spectroscopy (NIR-DCS) is an optical technique for estimating relative changes in skeletal muscle perfusion during exercise but may be affected by changes in cutaneous blood flow, as photons emitted by the laser must first pass through the skin. Accordingly, the purpose of this investigation was to examine how increased cutaneous blood flow affects NIR-DCS blood flow index (BFI) at rest and during exercise using a passive whole body heating protocol that increases cutaneous, but not skeletal muscle, perfusion in the uncovered limb. BFI and cutaneous perfusion (laser-Doppler flowmetry) were assessed in 15 healthy young subjects before (e.g., rest) and during 5 min of moderate-intensity handgrip exercise in normothermic conditions and after cutaneous blood flow was elevated via whole body heating. Hyperthermia significantly increased both cutaneous perfusion (∼7.3-fold; P ≤ 0.001) and NIR-DCS BFI (∼4.5-fold; P ≤ 0.001). Although relative BFI (i.e., fold-change above baseline) exhibited a typical exponential increase in muscle perfusion during normothermic exercise (2.81 ± 0.95), there was almost no change in BFI during hyperthermic exercise (1.43 ± 0.44). A subset of eight subjects were subsequently treated with intradermal injection of botulinum toxin-A (Botox) to block heating-induced elevations in cutaneous blood flow, which 1) nearly abolished the hyperthermia-induced increase in BFI and 2) restored BFI kinetics during hyperthermic exercise to values that were not different from normothermic exercise (P = 0.091). Collectively, our results demonstrate that cutaneous blood flow can have a substantial, detrimental impact on NIR-DCS estimates of skeletal muscle perfusion and highlight the need for technical and/or pharmacological advancements to overcome this issue moving forward.NEW & NOTEWORTHY 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.

Effects of old age and contraction mode on knee extensor muscle ATP flux and metabolic economy in vivo.

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. ABSTRACT: Muscle 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.

Rates of oxidative ATP synthesis are not augmented beyond the pH threshold in human vastus lateralis muscles during a stepwise contraction protocol.

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 (ATPCOST ) or an increased oxygen cost of ATP synthesis is unclear. We used 31 P-MRS to measure changes in cytosolic [ADP] (intramyocellular marker of oxidative metabolism), oxidative ATP synthesis (ATPOX ) and ATPCOST during a 6-stage, stepwise knee extension protocol. ATPCOST was unchanged across stages. The relationship between [ADP] and muscle power output was augmented at workloads above the pH threshold (pHT ; proxy for LT), whereas increases in ATPOX were attenuated. These results suggest the greater oxygen cost of contractions at workloads beyond the pHT is not caused by mechanisms that increase ATPCOST , but rather mechanisms that alter intrinsic mitochondrial function or capacity. ABSTRACT: 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 (ATPCOST ) or changes in the efficiency of mitochondrial oxygen consumption and oxidative ATP synthesis (ATPOX ). To partition these two hypotheses in vivo, we used 31 P-MRS to calculate slopes relating step-changes in muscle work to concurrent changes in cytosolic phosphates and ATPOX before and after the pH threshold (pHT ; 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 ATPOX were linearly related to workload below the pHT . However, slopes above the pHT were greater for muscle phosphates (P < 0.05) and lower for ATPOX (P < 0.05) than were the slopes observed below the pHT . The maximal capacity for ATPOX ( V̇max ) and ADP-specific ATPOX also declined beyond the pHT (P < 0.05), whereas ATPCOST was unchanged (P = 0.10). These results oppose the hypothesis that high-intensity contractions increase ATPCOST and suggest that greater oxidative metabolism at workloads beyond the pHT 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.

Impact of changes in tissue optical properties on near-infrared diffuse correlation spectroscopy measures of skeletal muscle blood flow.

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.

Muscle architecture, voluntary activation, and low-frequency fatigue do not explain the greater fatigue of older compared with young women during high-velocity contractions.

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.

Kinetic differences between macro- and microvascular measures of reactive hyperemia.

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.

Validity and accuracy of calculating oxidative ATP synthesis in vivo during high-intensity skeletal muscle contractions.

Several methods have been developed for using 31 P-MRS to calculate rates of oxidative ATP synthesis (ATPOX ) during muscular contractions based on assumptions that (1) the ATP cost of force generation (ATPCOST ) remains constant or (2) Michaelis-Menten coupling between cytosolic ADP and ATPOX does not change. However, growing evidence suggests that one, or both, of these assumptions are invalid during high-intensity fatigue protocols. Consequently, there is a need to examine the validity and accuracy of traditional ATPOX calculation methods under these conditions. To address this gap, we measured phosphate concentrations and pH in the vastus lateralis muscle of nine young adults during four rest-contraction-recovery trials lasting 24, 60, 120, and 240 s. The initial velocity of phosphocreatine resynthesis (ViPCr ) following each trial served as the criterion measure of ATPOX because this method makes no assumptions of constant ATPCOST or Michaelis-Menten coupling between changes in cytosolic ADP and ATPOX . Subsequently, we calculated ATPOX throughout the 240 s trial using several traditional calculation methods and compared estimations of ATPOX from each method with time-matched measurements of ViPCr . Method 1, which assumes that ATPCOST does not change, was able to model changes in ViPCr over time, but showed poor accuracy for predicting ViPCr across a wide range of ATPOX values. In contrast, Michaelis-Menten methods, which assume that the relationship between changes in cytosolic ADP and ATPOX remains constant, were invalid because they could not model the decline in ViPCr . However, adjusting these Michaelis-Menten methods for observed changes in maximal ATPOX capacity (i.e., Vmax ) permitted modeling of the decline in ViPCr and markedly improved accuracy. The results of these comprehensive analyses demonstrate that valid, accurate measurements of ATPOX can be obtained during high-intensity contractions by adjusting Michaelis-Menten ATPOX calculations for changes in Vmax observed from baseline to post-fatigue.

Magnetic Resonance Compatible Knee Extension Ergometer.

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 270 deg/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.

Oxidative ATP synthesis in human quadriceps declines during 4 minutes of maximal contractions.

KEY POINTS: During maximal exercise, skeletal muscle metabolism and oxygen consumption remain elevated despite precipitous declines in power. Presently, it is unclear whether these responses are caused by an increased ATP cost of force generation (ATPCOST ) or mitochondrial uncoupling; a process that reduces the efficiency of oxidative ATP synthesis (ATPOX ). To address this gap, we used 31-phosphorus magnetic resonance spectroscopy to measure changes in ATPCOST and ATPOX in human quadriceps during repeated trials of maximal intensity knee extensions lasting up to 4 min. ATPCOST remained unchanged. In contrast, ATPOX plateaued by ∼2 min and then declined (∼15%) over the final 2 min. The maximal capacity for ATPOX (Vmax ), as well as ADP-specific rates of ATPOX , were also significantly diminished. Collectively, these results suggest that mitochondrial uncoupling, and not increased ATPCOST , is responsible for altering the regulation of skeletal muscle metabolism and oxygen consumption during maximal exercise. ABSTRACT: The relationship between skeletal muscle oxygen consumption and power output is augmented during exercise at workloads above the lactate threshold. Potential mechanisms for this response have been hypothesized, including increased ATP cost of force generation (ATPCOST ) and mitochondrial uncoupling, a process that reduces the efficiency of oxidative ATP synthesis (ATPOX ). To test these hypotheses, we used phosphorus magnetic resonance spectroscopy to non-invasively measure changes in phosphate concentrations and pH in the vastus lateralis muscle of nine young adults during repeated trials of maximal, all-out dynamic knee extensions (120°s-1 , 1 every 2 s) lasting 24, 60, 120, and 240 s. ATPOX was measured at each time point from the initial velocity of PCr resynthesis, and ATPCOST was calculated as the sum of ATP synthesized by the creatine and adenylate kinase reactions, non-oxidative glycolysis, ATPOX and net changes in [ATP]. Power output declined in a reproducible manner for all four trials. ATPCOST did not change over time (main effect P = 0.45). ATPOX plateaued from 60 to 120 s and then decreased over the final 120 s (main effect P = 0.001). The maximal capacity for oxidative ATP synthesis (Vmax ), as well as ADP-specific rates of ATPOX , also decreased over time (main effect P = 0.001, both). Collectively, these results demonstrate that prolonged maximal contraction protocols impair oxidative energetics and implicate mitochondrial uncoupling as the mechanism for this response. The causes of mitochondrial uncoupling are presently unknown but may offer a potential explanation for the dissociation between skeletal muscle power output and oxygen consumption during maximal, all-out exercise protocols.