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Summary Objective To measure the variability of T 1ρ relaxation times using CubeQuant, T 2 relaxation times using quantitative double echo in steady state (DESS), and normalized sodium signals using 3D cones sodium magnetic resonance imaging (MRI) of knee cartilage in vivo at 3 T. Design Eight healthy subjects were scanned at 3 T at baseline, 1 day, 5 months, and 1 year. Ten regions of interest (ROIs) of knee cartilage were segmented in the medial and lateral compartments of each subject's knee. T 1ρ and T 2 relaxation times and normalized sodium signals were measured and the root-mean-square coefficient of variation (CV RMS ) was calculated. Intra-subject variability was measured over short, moderate and long-term, as well as intra-observer and inter-observer variability. Results The average intra-subject CV RMS measurements over short, moderate, and long-term time periods were 4.6%, 6.1%, and 6.0% for the T 1ρ measurements, 6.4%, 9.3%, and 10.7% for the T 2 measurements and 11.3%, 11.6%, and 12.9% for the sodium measurements, respectively. The average CV RMS measurements for intra-observer and inter-observer segmentation were 3.8% and 5.7% for the T 1ρ measurements, 4.7% and 6.7% for the T 2 measurements, and 8.1% and 11.4% for the sodium measurements, respectively. Conclusions These CV RMS measurements are substantially lower than previously measured changes expected in patients with advanced osteoarthritis compared to healthy volunteers, suggesting that CubeQuant T 1ρ , quantitative DESS T 2 and 3D cones sodium measurements are sufficiently sensitive for in vivo cartilage studies.