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[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MitoEAGLE|COST Action MitoEAGLE]] A wide diversity of substrate-uncoupler-inhibitor titration (SUIT) protocols has been used for evaluation of mitochondrial respiration in different samples and under various experimental conditions [1]. We developed a SUIT reference protocol (RP) with the goal to provide a common reference for establishing a database on comparative mitochondrial physiology [2]. The SUIT-RP consists of two complementary SUIT protocols: SUIT-RP1 [3] and SUIT-RP2 [4]. These protocols comprise specific respiratory pathways (FNSGp) converging at the Q-junction (Figure 1). RP1 and RP2 are harmonized by inclusion of two comparable respiratory states (SGp_''E'' and CIV_''E''). Harmonized states differ in terms of the sequence of substrate, uncoupler and inhibitor titrations (Figure 2). RP1 includes respiratory states that can be compared to many previously published SUIT protocols. In the present communication, we address specifically the following topics: Are ET capacities identical in the harmonized states of RP1 and RP2 applied in parallel experiments? In our updated ‘final’ version of RP2, the sequence and concentrations of malate titrations was optimized to distinguish fatty acid oxidation capacity from the N-pathway capacity. The harmonized respiratory states were in excellent agreement in a parallel experimental design with RP1 and RP2 (test and ‘final’) applied to (i) isolated mouse cardiac mitochondria, (ii) brain tissue homogenate from mice, and (iii) cryopreserved permeabilized HEK cells [5]. On the other hand, previous studies demonstrate [6] that N pathway cannot be fully isolated from S pathway. S-contribution in SUIT protocols depends on the N-substrate combinations used. We tested S-contribution in RP1 and RP2 protocols for a better understanding of bioenergetic complexity in mitochondrial physiology. In conclusion, an optimal design and comprehension of the SUIT protocols is crucial for a proper evaluation of mitochondrial function in healthy and pathological conditions.