Background: An Achilles tendon rupture (ATR) is a frequent injury and results in the activation of tendon cells and collagen expression, but it is unknown to what extent turnover of the tendon matrix is altered before or after a rupture. Purpose/Hypothesis: The purpose of this study was to characterize tendon tissue turnover before and immediately after an acute rupture in patients. It was hypothesized that a rupture would result in pronounced collagen synthesis in the early phase (first 2 weeks) after the injury. Study Design: Cross-sectional study; Level of evidence, 3. Methods: The study included patients (N = 18) eligible for surgery after an ATR. At the time of inclusion, the patients ingested deuterium oxide (²H₂O) orally, and on the day of surgery (within 14 days of the injury), they received a 3-hour flood-primed infusion of an ¹⁵N-proline tracer. During surgery, the patients had 1 biopsy specimen taken from the ruptured part of the Achilles tendon and 1 that was 3 to 5 cm proximal to the rupture as a control. The biopsy specimens were analyzed for carbon-14 (¹⁴C) levels in the tissue to calculate long-term turnover (years), incorporation of ²H-alanine (from ²H₂O) into the tissue to calculate the fractional synthesis rate (FSR) of proteins in the short term (days), and incorporation of ¹⁵N-proline into the tissue to calculate the acute FSR (hours). Results: Both the rupture and the control samples showed consistently lower levels of ¹⁴C compared with the predicted level of ¹⁴C in a healthy tendon, which indicated increased tendon turnover in a fraction (48% newly synthesized) of the Achilles tendon already for a prolonged period before the rupture. Over the first days after the rupture, the synthesis rate for collagen was relatively constant, and the average synthesis rate on the day of surgery (2-14 days after the rupture) was 0.025% per hour, irrespective of the length of time after a rupture and the site of sampling (rupture vs control). No differences were found in the FSR between the rupture and control samples in the days after the rupture. Conclusion: Higher than normal tissue turnover in the Achilles tendon before a rupture indicated that changes in the tendon tissue preceded the injury. In addition, we observed no increase in tendon collagen tissue turnover in the first 2 weeks after an ATR. This favors the view that an increase in the formation of new tendon collagen is not an immediate phenomenon during the regeneration of ruptured tendons in patients. Registration: NCT03931486 (ClinicalTrials.gov identifier).