Dynamic changes in circulating and tissue metabolites and lipids occur in response to exercise-induced cellular and whole-body energy demands to maintain metabolic homeostasis. The metabolome and lipidome in a given biological system provides a molecular snapshot of these rapid and complex metabolic perturbations. The application of metabolomics and lipidomics to map the metabolic responses to an acute bout of aerobic/endurance or resistance exercise has dramatically expanded over the past decade thanks to major analytical advancements, with most exercise-related studies to date focused on analyzing human biofluids and tissues. Experimental and analytical considerations, as well as complementary studies using animal model systems, are warranted to help overcome challenges associated with large human interindividual variability and decipher the breadth of molecular mechanisms underlying the metabolic health-promoting effects of exercise. In this review, we provide a guide for exercise researchers regarding analytical techniques and experimental workflows commonly used in metabolomics and lipidomics. Furthermore, we discuss advancements in human and mammalian exercise research utilizing metabolomic and lipidomic approaches in the last decade, as well as highlight key technical considerations and remaining knowledge gaps to continue expanding the molecular landscape of exercise biology.

Quadriceps muscle deficits following anterior cruciate ligament (ACL) injury can persist years beyond reconstruction (ACLR), but the underlying molecular mechanisms remain largely unexplored. This study investigated whether persistent muscle deficits following ACLR are associated with defects in hypertrophic signaling pathways in response to acute resistance exercise. Twelve participants (7 females, 5 males; age 26.7 ± 4.1 years; 2.6 ± 1.5 years post‐ACL injury) underwent MRI scanning and one‐repetition maximum (1RM) testing to determine muscle volume and unilateral leg‐press strength in the ACLR versus contralateral control leg. Following a standardized meal and overnight fast, participants performed unilateral leg‐press resistance exercise (6 sets of 8 repetitions at 80% 1RM). Vastus lateralis (VL) muscle biopsies were collected from both legs at rest and immediately post‐exercise. Immunoblotting assessed phosphorylation status and total protein content of hypertrophic signaling proteins (e.g., mTORC1 pathway). Total knee extensor muscle volume (7.5% deficit; p = 0.037), VL (7.7% deficit; p = 0.031), and rectus femoris (5.8% deficit; p = 0.022) muscle volumes were decreased in the ACLR versus contralateral control leg. Knee extensor ( R ² = 0.37; p = 0.036) and VL ( R ² = 0.36; p = 0.038) deficits correlated with increased time since injury, with no associated unilateral leg press 1RM strength difference between legs ( p = 0.192). Despite main effects of exercise observed for mTORC1 pathway signaling proteins (i.e., decreased p‐TSC2 Thr1462; p = 0.002 and p‐4E‐BP1 Thr37/46; p < 0.0001, and increased p‐S6 Ser240/244; p = 0.032), no between‐leg differences in mTORC1 signaling were detected at rest or immediately post‐exercise under the specific experimental conditions tested. In conclusion, persistent muscle volume deficits following ACLR were less pronounced with increased time since injury but were not associated with detectable differences between legs in unilateral leg‐press strength. The lack of detectable between‐leg differences in vastus lateralis mTORC1 signaling warrants further investigation at timepoints closer to ACLR and/or later post‐exercise. Trial Registration: This trial was prospectively registered at the Australian New Zealand Clinical Trials Registry (ANZCTR; trial registration number 12622001166763)

Background/Objectives: Acute exercise remodels many interconnected biochemical pathways in metabolically active tissues. This remodeling involves the activation of the energy-sensing AMP-activated protein kinase (AMPK) to maintain cellular energy homeostasis. Critical energy reserves of glycogen, primarily stored in liver and skeletal muscle and known to interact with AMPK, are utilized to help meet increased energy demands with exercise. However, the breadth of metabolic pathways regulated by acute exercise and AMPK’s interactive roles with glycogen remain incompletely understood. This study therefore aimed to map mouse liver and skeletal muscle metabolite responses to continuous acute exercise and disruption of AMPK-glycogen interactions. Methods: Liquid chromatography–mass spectrometry-based untargeted metabolomics was used to measure the relative abundance of liver and gastrocnemius muscle metabolites at rest and following an acute bout of continuous treadmill running in wild type (WT) and AMPK transgenic mice with double knock-in (DKI) mutations in the β subunit carbohydrate binding module that mediates glycogen binding. Results: Over 200 total metabolites were identified/annotated across liver and skeletal muscle, including 45 metabolites responsive to exercise (p < 0.05; FDR < 0.1). Exercise-regulated metabolites included known metabolic pathways and metabolites never associated or with only emerging evidence related to exercise (e.g., ergothioneine) and/or AMPK-glycogen interactions (N6,N6,N6-trimethyl-L-lysine, a precursor of L-carnitine). Conclusions: Liver and skeletal muscle metabolomic profiles displayed shifts between WT and DKI mice at rest, with shifts also detected following a continuous acute exercise bout. An interaction effect was also observed in skeletal muscle, suggesting differential muscle metabolite responses to acute exercise in DKI mice that may contribute to their functional impairments in metabolic control and exercise capacity versus WT. Collectively, these findings expand the molecular landscape of acute exercise and reveal liver and muscle metabolites underlying exercise-induced metabolic responses.