Quantitative T1rho magnetic resonance imaging (MRI) can potentially help identify early-stage osteoarthritis (OA) by non-invasively assessing proteoglycan concentration in articular cartilage. T1rho relaxation times are negatively correlated with proteoglycan concentration. Cartilage compresses in response to load, resulting in water exudation, a relative increase in proteoglycan concentration, and a decrease in the corresponding T1rho relaxation times. To date, there is limited information on changes in cartilage composition resulting from daily activity. Therefore, the objective of this study was to quantify changes in tibial cartilage T1rho relaxation times in healthy human subjects following activities of daily living. It was hypothesized that water exudation throughout the day would lead to decreased T1rho relaxation times. Subjects underwent MR imaging in the morning and afternoon on the same day and were free to go about their normal activities between scans. Our findings confirmed the hypothesis that tibial cartilage T1rho relaxation times significantly decreased (by 7%) over the course of the day with loading, which is indicative of a relative increase in proteoglycan concentration. Additionally, baseline T1rho values varied with position within the cartilage, supporting a need for site-specific measurements of T1rho relaxation times. Understanding how loading alters the proteoglycan concentration in healthy cartilage may hold clinical significance pertaining to cartilage homeostasis and potentially help to elucidate a mechanism for OA development. These results also indicate that future studies using T1rho relaxation times as an indicator of cartilage health should control the loading history prior to image acquisition to ensure the appropriate interpretation of the data.