The study focused on fifty community-dwelling adults aged 65 to 85, twenty-two men and twenty-eight women, who were recreationally active but not engaged in structured exercise more than twice per week. All participants had body mass indices between 22 and 30 kilograms per square metre, were non-smokers, and were free of severe liver, kidney, rheumatic or malignant disease. Recruited through community advertisements between December 2023 and March 2025, they arrived at the laboratory early in the morning after an overnight fast, where researchers measured their body composition, muscle strength, functional performance, and substrate utilization during exercise, alongside the mitochondrial respiration of freshly isolated blood cells.

The measurement of mitochondrial function in PBMCs relied on high-resolution respirometry performed with an OROBOROS Oxygraph-2k at 37 degrees Celsius. Four million viable cells, isolated through density gradient centrifugation within three hours of blood collection, were suspended in respiration buffer and subjected to a sequence of pharmacological perturbations. First, ROUTINE respiration, the basal oxygen consumption reflecting physiological ATP turnover in intact cells, was recorded. Oligomycin A was then added to inhibit ATP synthase, revealing LEAK respiration, the non-phosphoryiating oxygen consumption driven by proton leak across the inner mitochondrial membrane. Titration of the uncoupler FCCP forced the electron transport system to its theoretical ceiling, yielding MAX respiration, and finally rotenone and antimycin A shut down the respiratory chain entirely to quantify non-mitochondrial oxygen consumption. All oxygen fluxes were corrected for this residual background and expressed relative to cell count, and derived parameters included reserve capacity, the difference between MAX and ROUTINE respiration, and ATP-linked respiration, the difference between ROUTINE and LEAK.

To assess metabolic flexibility, the researchers evaluated substrate utilization during a ten-minute treadmill walk at approximately 60 percent of each participant’s estimated VO2max, performed after a standardized 345-kilocalorie test meal containing 56 grams of carbohydrate, 9.2 grams of fat, and 6.9 grams of protein. Breath-by-breath gas exchange was recorded with a calibrated metabolic cart during the final steady-state minutes, and the respiratory exchange ratio, the ratio of carbon dioxide production to oxygen consumption, was averaged over the last two minutes. Fat and carbohydrate oxidation rates were calculated from these gas exchange values using the non-protein stoichiometric equations described by Frayn. Lower RER and greater reliance on fat during moderate exercise were interpreted as markers of greater metabolic flexibility, since fat is typically the predominant fuel at rest and at intensities up to roughly 65 percent of VO2max, while carbohydrate oxidation rises as intensity increases.

The results showed a clear pattern. ROUTINE respiration was negatively correlated with RER and with carbohydrate utilization, and positively correlated with fat utilization, and ATP-linked respiration displayed the same relationships. These associations held firm in linear regression models adjusted for age, sex, skeletal muscle index, habitual physical activity level, and high-sensitivity C-reactive protein, suggesting that the link between basal mitochondrial activity in blood cells and whole-body substrate preference is independent of these confounders. Notably, LEAK and MAX respiration did not correlate with any measure of metabolic flexibility, hinting that endogenous, unstressed ATP turnover in immune cells is more informative about everyday energy metabolism than the stress-induced respiratory states that many bioenergetics studies emphasize.

Physical performance told a parallel story. Higher ROUTINE respiration was associated with stronger handgrip and faster gait speed over a four-metre walk, with the gait speed association remaining significant after adjustment. ATP-linked respiration also correlated with gait speed. Intriguingly, LEAK respiration, often regarded as an inefficiency because it consumes oxygen without producing ATP, was positively associated with one-repetition maximum quadriceps strength on a leg extension machine, pointing to a possible role of proton leak-related respiration in lower-body muscular capacity. Maximal respiratory capacity, by contrast, was not associated with any performance measure, which the authors attribute to the submaximal nature of the exercise protocol: a theoretical ceiling that is rarely approached under ordinary physiological conditions would not be expected to track everyday function.

To probe whether distinct mitochondrial phenotypes exist among older adults, the team applied unsupervised k-means clustering to log-transformed ROUTINE, LEAK, and MAX values. Two clusters emerged. Participants in the high-respiration cluster showed ROUTINE respiration of 3.72 versus 2.10 picomoles of oxygen per second per million cells, LEAK respiration of 1.35 versus 0.61, and MAX respiration of 7.78 versus 5.46 compared with the low-respiration group, all differences highly significant. The high-respiration group also burned fat more readily during the treadmill test, with a lower RER of 0.88 versus 0.89 and fat utilization of 39.7 versus 35.8 percent of energy expenditure, consistent with greater metabolic flexibility. Logistic regression further revealed that higher countermovement jump height and greater quadriceps strength were associated with lower odds of belonging to the low-respiration cluster. One unexpected observation was that the high-respiration group carried higher concentrations of C-reactive protein, a systemic inflammatory marker, and the authors note that inflammatory-driven shifts in PBMC subpopulations, particularly monocytes, may have influenced respiratory profiles even after statistical adjustment.

The study extends a growing body of evidence that blood-cell bioenergetics reflect systemic physiology. Previous work had linked PBMC respiratory capacity to age-related fatigue, type 2 diabetes, cardiovascular disease, and obesity, and prior studies by other groups found that higher maximal PBMC respiration tracked with gait speed and lower-extremity strength in overweight and obese older adults. The new research pushes these associations into a healthier, community-dwelling population and connects them, for the first time, to a standardized assessment of exercise substrate utilization. The findings resonate with observations from the Baltimore Longitudinal Study of Aging, in which higher mitochondrial respiration clustered with skeletal muscle oxidative capacity and in vivo phosphorus-31 magnetic resonance spectroscopy markers of mitochondrial health. Together, they support the idea that mitochondrial respiratory capacity, recognized as a hallmark of ageing, is not merely a tissue-specific phenomenon but an integrative property that links muscle energetics, immune cell metabolism, and whole-body physiological function.

The authors are careful to acknowledge the limits of the cross-sectional design, which precludes causal claims about whether robust mitochondria preserve metabolic flexibility and strength or vice versa. PBMCs, while minimally invasive and accessible, comprise a heterogeneous mixture of monocytes and lymphocytes whose bioenergetic profiles differ, and they cannot fully capture the respiratory capacity of skeletal muscle mitochondria, the tissue most directly responsible for whole-body substrate oxidation. Metabolic flexibility was also assessed during a single fixed-intensity, postprandial exercise bout rather than across a dynamic range of nutritional states, and the healthy, recreationally active sample limits generalizability to frail or metabolically compromised populations. Fasted versus fed comparisons and longitudinal follow-up would help clarify whether blood-cell respiration can serve as a predictive biomarker rather than a snapshot correlate.

Even so, the implications are compelling. If a routine blood test measuring PBMC mitochondrial respiration can flag older adults with declining metabolic flexibility or emerging functional limitations, clinicians could intervene earlier with exercise training, dietary strategies, or other approaches known to improve mitochondrial function and preserve independence. As populations age worldwide, the search for accessible biomarkers of biological ageing has intensified, and this study offers a technically straightforward candidate: a measure of how efficiently the mitochondria inside a person’s immune cells convert oxygen into energy, and how much reserve capacity they retain. The researchers conclude that mitochondrial respiratory capacity in PBMCs is associated with both substrate utilization during standardized postprandial submaximal exercise and key markers of physical performance in healthy older adults, and that a high-respiration phenotype, identifiable by unsupervised clustering, coincides with a more favourable metabolic profile and preserved quadriceps strength and function. What circulates in our blood, it appears, tells a story about how our bodies move, burn fuel, and endure.