Neuropeptide Y (NPY) is a 36-amino acid peptide circulating at a subpicomolar concentration participating in multiple physiological and pathological processes. NPY is prone to peptidolysis, generating metabolites with modified affinity for the five known receptors of NPY that mediate distinct effects. It is, therefore, crucial to distinguish each metabolite to understand the multiple functions of NPY. Since immunoassays are not able to distinguish NPY from its metabolites, we have validated a microliquid chromatography tandem mass spectrometry (micro-LC-MS/MS) assay for the quantification of endogenous NPY, NPY2-36, NPY3-36, NPY1-35, and NPY3-35 in human plasma. Sample preparation relies on immunoextraction in 96-well plates, followed by solid-phase extraction prior to micro-LC-MS/MS. The LLOQ ranged from 0.03 to 0.16 pM, intraand inter-assay precision were <27% and trueness <22%. We determined reference intervals in 155 healthy volunteers and 40 hypertensive patients. We found that NPY3-36 is the main circulating peptide in resting conditions and that NPY and catecholamines are simultaneously increased during orthostasis. We also showed that the concentrations of NPY and its metabolites are similar in healthy volunteers and hypertensive patients. NPY is the prototype peptide that circulates in concentrations expected to be beyond instrumental capacities. We have been successful in developing a high-throughput specific and sensitive assay by including a deep knowledge of the physicochemical properties of these peptides to an efficient multistep sample preparation, and a *

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Neuropeptide Ys (NPYs) contribute to sympathetic‐adreno stimulation: NPY1‐36 potentiates the effects of catecholamines (CATs), whereas NPY3‐36 inhibits CAT release. We sought to investigate whether inhibiting dipeptidyl‐peptidase‐4 (DPP4), cleaving NPY1‐36 into NPY3‐36, leads to increased NPY1‐36 potentiating effects and reduced NPY3‐36 inhibitory effects on CATs, thereby improving endurance performance. Seven male participants (age 27 ± 3 years, BMI 23.1 ± 2.4 kg/m 2 ) performed time‐to‐exhaustion cycling exercise at 95% of peak power output with either placebo, or saxagliptin, a DPP4 inhibitor. Oxygen consumption (V̇O 2 ), heart rate variability, NPY1‐36, NPY3‐36, catecholamines, and lactate were measured at several time points before, during, and after exercise. With saxagliptin, DPP4 activity (12.7 ± 1.6 vs. 0.2 ± 0.3 U/L, p = 0.001; d = 10.7) was decreased at rest, while NPY3‐36 (1.94 ± 0.88 vs. 0.73 ± 0.22 pm ; p < 0.001; d = 2.04) decreased and NPY1‐36 increased during exercise (2.64 ± 2.22 vs. 4.59 ± 2.98 pm ; p < 0.01; d = 0.19). CATs were unchanged. Time‐to‐exhaustion was 32% higher with saxagliptin. The difference in time‐to‐exhaustion between placebo and saxagliptin was correlated with NPY1‐36 differences (R = 0.78, p < 0.05). Peak V̇O 2 and other cardio‐respiratory values were not different, whereas peak NPY concentrations were higher with saxagliptin. DPP4 blockade improved performance, increased NPY1‐36, and decreased NPY3‐36 concentrations which may have potentiating effects on the influences of CATs. However, DPP4 is involved in many different actions, thus NPYs are one group of factors that may underly its performance‐enhancing effects; further studies are required to determine the exact mechanisms.