The ability to measure lactate in vivo is valuable in the study of many pathologies where metabolic regulation is compromised, including cancer, cardiac failure, diabetes, and other metabolic disorders. Traditional magnetic resonance spectroscopy (¹H and ¹³C MRS)¹ of lactate is limited in vivo by spectral overlap with lipids or inadequate sensitivity and/or poor spatial resolution. Fast kinetics (<1min) of lactate metabolism can be studied with high sensitivity using dynamic nuclear polarization (DNP)² but requires special polarizing equipment and complex modeling for data analysis. In this study, we used chemical exchange saturation transfer (CEST)³ to exploit the exchange of –OH protons on lactate with bulk water (LATEST). This method was validated in vivo with a human exercise study where an increase in lactate CEST was measured after intense exercise. With CEST, lactate can be imaged with high sensitivity and spatial resolution, and is not limited by time constraints of hyperpolarized imaging.All human studies were approved by the Institutional Review Board protocol of the University of Pennsylvania. Informed consent was obtained from all subjects prior to imaging. Human skeletal muscle studies were performed on a whole-body 7T whole-body MRI scanner (Siemens, Erlangen, Germany) with a 28-channel QED knee RF coil. LATEST imaging was performed on the right calf muscle of 5 healthy male subjects (ages 21-35). CEST images and WASSR⁴ B₀ and B₁ field maps were acquired while the subjects rested. CEST images were collected using a saturation pulse consisting of a 3s long saturation pulse train of 30 100ms long Hanning windowed saturation pulses (B_1rms = 0.73μT). Imaging parameters: slice thickness =10mm, SHOT TR=6s, GRETR=6.1 ms, TE=2.9ms, FOV = 140x140 mm², matrix size 128x128. CEST images were acquired from 0 - ±0.8 ppm, in steps of 0.1ppm. After baseline image acquisition, subjects were instructed to perform plantar flexion exercise in-magnet with an MR-compatible, pneumatically-controlled foot pedal, until exhaustion. Immediately following exercise, CEST images were acquired with a time resolution of 1.8 minutes. B₁ and WASSR maps were acquired again after post-exercise CEST images. Lactate-edited spectroscopy was also performed the right calf muscle of three healthy male subjects (ages 24-65). In order to select a voxel in the desired muscle region, an inversion spectroscopy (ISIS) localization⁵ was implemented before a selective multiple quantum coherence (MCQ) editing sequence⁶. For all three subjects, a voxel of 30x80x40mm was placed over the calf muscle including the gastrocnemius muscles and a portion of the soleus muscle. Spectra were first acquired for 96s while the subject rested. The same exercise paradigm was repeated as described above. After exercise, spectra were acquired for a total of 20 minutes. For quantification, water spectra and lactate spectra were acquired from the same voxel. Spectroscopy parameters: TR=3s, TE=165.6ms, dummy scans=4, water averages=8, lactate edited spectra averages=8*n, n=2 for pre-exercise, n=50 for post-exercise.During resting state imaging, minimal LATEST signal was observed in human skeletal muscle (Figure 1a-b). After intense anaerobic exercise, when glycolysis leads to production of lactate, an increase of ~8% CESTasym was measured in the gastrocnemius muscle (Figure 1c). After ~18 minutes minimal LATEST signal is detected as the lactate recovers to baseline (Figure 1d). Immediately after exercise, lactate asymmetry peaked at ~0.4ppm downfield from bulk water, with a B_1rms of 0.73uT and a saturation duration of 3s as shown in the medial gastrocnemius (Figure 2). Recovery of lactate levels to baseline over ~18 minutes (Figure 3) from 5 subjects’ medial gastrocnemius muscle corresponds with recovery measured by lactate-edited spectroscopy (Figure 4). Correlation between spectroscopy-derived lactate and CEST-derived lactate concentration (Figure 5) gives a slope of ~0.56% per mM of lactate from spectroscopy. These results are consistent with reported lactate concentration increase measured in muscle biopsy after intense exercise⁷. We have demonstrated that LATEST can be used to measure in vivo, dynamic changes in lactate with high sensitivity and spatial resolution. Lactate levels are often increased as a result of metabolic impairment. This technique can be applied to monitor therapeutic response in a wide range of disorders where lactate metabolism is implicated, including genetic diseases with inborn errors of metabolism, and cancer.