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Creatine kinase catalyses reversible transfer between phosphocreatine and ADP to yield creatine and ATP. Inferring free ADP from this reaction requires an appropriate equilibrium model and information about pH, magnesium and the creatine pool.

In vivo muscle NMR reveals energy-buffer use, phosphate chemistry and recovery kinetics without repeatedly removing tissue. It measures visible metabolites, not metabolic flux automatically. A particularly informative experiment is localized ${}^{31}\mathrm{P}$ nuclear magnetic resonance, with a limb positioned in a magnet and a radiofrequency coil over the exercising muscle. Repeated spectra can be collected at rest, during a defined workload and throughout recovery. Localization and synchronized contraction reduce motion artefacts and contamination from inactive tissue.

The resting ${}^{31}\mathrm{P}$ spectrum resolves phosphocreatine, inorganic phosphate and the phosphate resonances of ATP, together with some other phosphorus-containing metabolites. Peak areas, after correcting for excitation and relaxation effects, report relative quantities of NMR-visible pools. An external reference or another justified calibration is needed for absolute concentrations. Large phosphocreatine stores relative to ATP illustrate a buffering system, but a resting spectrum alone does not reveal how fast ATP is synthesized or consumed.

During exercise, creatine kinase buffers ATP throughConsequently phosphocreatine often falls and inorganic phosphate rises while ATP remains comparatively stable during moderate exercise. Stable ATP does not mean little ATP turnover: it can mean rapid replenishment matching rapid consumption. Serial spectra relate changes in the phosphocreatine-to-phosphate ratio to workload and fatigue. Gated human-muscle experiments directly demonstrated such workload-dependent energetic responses.^[ref]

The chemical shift of inorganic phosphate relative to phosphocreatine depends on its protonation state and therefore permits an estimate of intracellular pH. With appropriate calibrated acid and base endpoint shifts, a commonly used relation has the formThe observed shift must lie between the calibration endpoints, and the relevant calibration depends on experimental conditions. Exercise-related acidification and its recovery can therefore be followed alongside the energy-buffer changes. A resolved pair of phosphate signals may indicate metabolically distinct compartments; fitting one average shift to a mixed signal can conceal heterogeneity. A pH fall is not simply a direct lactate measurement, because proton production, consumption, buffering and export all contribute.

If creatine kinase is close to equilibrium, measured ATP and phosphocreatine, together with estimates of total creatine, pH and free magnesium, can constrain free cytosolic ADP. This is an inference from an equilibrium model rather than a direct ADP peak measurement. The corresponding ATP hydrolysis free energy depends on metabolite activities through $\Delta G=\Delta G^{\circ \prime }+RT\ln ([\mathrm{ADP}][P_i]/[\mathrm{ATP}])$ at the specified biochemical conditions. Concentration ratios and a consistent standard-state convention are essential when using this expression quantitatively.

After exercise, oxidative ATP production replenishes phosphocreatine. Recovery is often summarized by $\mathrm{PCr}(t)={\mathrm{PCr}}_{\mathrm{end}}+\Delta \mathrm{PCr}(1-e^{-t/\tau })$, making the initial recovery rate $\Delta \mathrm{PCr}/\tau$. With adequate oxygen delivery and suitable pH conditions, recovery kinetics provide an index of oxidative capacity. Slow recovery may instead reflect impaired perfusion, severe acidification or continuing ATP demand, so it does not uniquely identify a mitochondrial enzyme defect. Work-matched comparisons, adequate rest, reproducible exercise and independent perfusion or oxygenation measurements strengthen interpretation.

Related experiments extend the information available. ${}^1\mathrm{H}$ spectroscopy can detect additional metabolites or lipid pools, while ${}^{13}\mathrm{C}$ experiments can follow labelled substrate incorporation or glycogen under suitable sensitivity conditions. Magnetization-transfer experiments can probe exchange, but exchange rates need not equal net ATP-production flux. The strength of the method is the time-resolved connection between muscle function and intracellular chemistry, with explicit limits from spatial averaging, sensitivity, calibration and the biochemical model used to interpret each signal.

The enzyme adenylate kinase rapidly interconverts the adenine nucleotides:For approximately fixed conditions and near-equilibrium activity ratios,The AMP:ATP ratio responds quadratically to ADP:ATP. Doubling ADP:ATP therefore quadruples AMP:ATP. In working skeletal muscle, ATP is also buffered by creatine kinase and continuing ATP production, so its fractional change is relatively small. The initially smaller AMP pool can undergo a much larger fractional increase, providing a sensitive signal of energetic stress to regulators such as AMP-activated protein kinase. This concerns sensitivity of relative changes; it does not mean that the absolute AMP concentration exceeds ATP.