Purpose The recovery of muscle oxygen consumption (m O 2 ) after exercise provides a measure of skeletal muscle mitochondrial capacity, as more and better-functioning mitochondria will be able to restore m O 2 faster to the pre-exercise state. The aim was to measure muscle mitochondrial capacity using near-infrared spectroscopy (NIRS) within a healthy, normally active population and relate this to parameters of aerobic fitness, investigating the applicability and relevance of using NIRS to assess muscle mitochondrial capacity non-invasively. Methods Mitochondrial capacity was analysed in the gastrocnemius and flexor digitorum superficialis (FDS) muscles of eight relatively high-aerobic fitness ( O 2 peak ≥ 57 mL/kg/min) and eight relatively low-aerobic fitness male subjects ( O 2 peak ≤ 47 mL/kg/min). Recovery of whole body O 2 , i.e. excess post-exercise oxygen consumption (EPOC) was analysed after a cycling protocol. Results Mitochondrial capacity, as analysed using NIRS, was significantly higher in high-fitness individuals compared to low-fitness individuals in the gastrocnemius, but not in the FDS ( p = 0.0036 and p = 0.20, respectively). Mitochondrial capacity in the gastrocnemius was significantly correlated with O 2 peak ( R 2 = 0.57, p = 0.0019). Whole body O 2 recovery was significantly faster in the high-fitness individuals ( p = 0.0048), and correlated significantly with mitochondrial capacity in the gastrocnemius ( R 2 = 0.34, p = 0.028). Conclusion NIRS measurements can be used to assess differences in mitochondrial muscle oxygen consumption within a relatively normal, healthy population. Furthermore, mitochondrial capacity correlated with parameters of aerobic fitness ( O 2 peak and EPOC), emphasising the physiological relevance of the NIRS measurements.

Mitochondrial capacity is pivotal to skeletal muscle function and is suggested to decline with age. However, there is large heterogeneity in current data, possibly due to effect modifiers such as physical activity, sex and muscle group. Yet, few studies have compared multiple muscle groups in different age groups with comparable physical activity levels. Here, we newly used near-infrared spectroscopy (NIRS) to characterise mitochondrial capacity in three different locomotor muscles in young (19-25 year) and older (65-71 year), healthy males with similar physical activity levels. Mitochondrial capacity and reperfusion after arterial occlusion was measured in the vastus lateralis (VL), the gastrocnemius (GA) and the tibialis anterior (TA). Physical activity was verified using accelerometry and was not different between the age groups (404.3 ± 214.9 vs 494.9 ± 187.0 activity kcal per day, p = 0.16). Mitochondrial capacity was significantly lower in older males in the GA and VL, but not in the TA (p = 0.048, p = 0.036 and p = 0.64, respectively). Reperfusion rate was not significantly different for the GA (p = 0.55), but was significantly faster in the TA and VL in the young group compared to the older group (p = 0.0094 and p = 0.039, respectively). In conclusion, we identified distinct modes of mitochondrial ageing in different locomotor muscles in a young and older population with similar physical activity patterns. Furthermore, we show that NIRS is suitable for relatively easy application in ageing research and can reveal novel insights into mitochondrial functioning with age.

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Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) provides key opportunities to study the pathophysiology of several diseases, such as type 2 diabetes, obesity and cancer. Extracellular flux (XF) assays provide dynamic metabolic analysis of living cells that can capture ex vivo cellular metabolic responses to biological stressors. To obtain reliable data from PBMCs from individuals, novel methods are needed that allow for standardization and take into account the non-adherent and highly dynamic nature of PBMCs. We developed a novel method for extracellular flux analysis of PBMCs, where we combined brightfield imaging with metabolic flux analysis and data integration in R. Multiple buffy coat donors were used to demonstrate assay linearity with low levels of variation. Our method allowed for accurate and precise estimation of XF assay parameters by reducing the standard score and standard score interquartile range of PBMC basal oxygen consumption rate and glycolytic rate. We applied our method to freshly isolated PBMCs from sixteen healthy subjects and demonstrated that our method reduced the coefficient of variation in group mean basal oxygen consumption rate and basal glycolytic rate, thereby decreasing the variation between PBMC donors. Our novel brightfield image procedure is a robust, sensitive and practical normalization method to reliably measure, compare and extrapolate XF assay data using PBMCs, thereby increasing the relevance for PBMCs as marker tissue in future clinical and biological studies, and enabling the use of primary blood cells instead of immortalized cell lines for immunometabolic experiments.