OBJECTIVES/GOALS: This project aimed to investigate differences between isometric and isotonic systems for low and high frequency contractions. Intracellular changes in pH caused by the rate of exercise differ in their effect on PCr. The project assessed the reproducibility between these exercise methods. METHODS/STUDY POPULATION: Four subjects were studied (2F/2M, avg. age 27 years). 31P-MRS spectra were measured in the vastus lateralis (VL) using a 3T MRI scanner using a 31P/1H surface coil. In the isometric studies, the (supine) subjects legs were supported by a wedge pad and restrained by straps over the hips and knee with a resistance strap over one ankle. In the isometric studies, the (prone) subjects legs were fit into machine designed to induce isometric exercise. There were 4 exercise bouts that differed by exercise type and frequency and, consequently, duration. 31P-MR spectra were acquired from a 25mm slab of leg positioned parasagittal along the VL. Analyses were done using jMRUI and R. RESULTS/ANTICIPATED RESULTS: With an increase in sample size, we expect differences between recovery time constants, tau, of PCr between the different exercise frequency groups alluded to by other studies investigating recovery rates in bicep skeletal muscle. From the preliminary data, we obtained large limits of agreement of tau = 0.053 s-1 & tau =-0.074 s-1 from using Bland-Altman analysis. As we increase our sample size, we anticipate that we will see differences of PCr recovery rates between the exercise types as well. DISCUSSION/SIGNIFICANCE: Current studies that investigate the differences in PCr recovery rates between different metabolic states differ in the exercise methods employed on subjects. This study aims to standardize PCr recovery assessment in the VL for more interpretable and applicable assessments of metabolic function.