Background: Physical activity (PA) is typically lower on hemodialysis (HD) days. Albeit intradialytic inactivity is expected, it is unknown whether recovery after HD contributes to low PA. We investigated the impact of HD and post-HD period on granular PA relative to HD timing. Methods: We used baseline data from the HDFIT trial conducted from August 2016 to October 2017. Accelerometry measured PA over one week in patients who received thrice-weekly high-flux HD (vintage 3 to 24 months), were clinically stable, and had no ambulatory limitations. PA was assessed on HD days (0 to ≤24 hours after start HD), first non-HD days (>24 to ≤48 hours after start HD) and second non-HD day (>48 to ≤72 hours after start HD). PA was recorded in blocks/slices: 4 hours during HD, 0 to ≤2 hours post-HD (30 minute slices), and >2 to ≤20 hours post-HD (4.5 hour slices). Blocks/slices of PA were captured at concurrent/parallel times on first/second non-HD days compared to HD days. Results: Among 195 patients (mean age 53±15 years, 71% male), step counts per 24-hours were 3919±2899 on HD days, 5308±3131 on first non-HD days (p<0.001), and 4926±3413 on second non-HD days (p=0.032). During concurrent/parallel times to HD on first and second non-HD days, patients took 1308 and 1128 more steps (both p<0.001). Patients took 276 more steps and had highest rates of steps/hour 2-hours post-HD versus same times on first non-HD days (all p<0.05). Consistent findings were observed on second non-HD days. Conclusions: PA was higher within 2-hours of HD versus same times on non-HD days. Lower PA on HD days was attributable to intradialytic inactivity. The established PA profiles are of importance to the design and development of exercise programs that aim to increase activity during and between HD treatments. Trial Registration: HDFIT was registered 20 April 2016 on ClinicalTrials.gov (NCT02787161) https://clinicaltrials.gov/ct2/show/NCT02787161.