Abstract: High-temperature waterlogging in summer is a significant environmental stress that affects alfalfa (Medicago sativa L.) growth in northern China. This study investigated the physiological and biochemical responses and quality changes in alfalfa to summer high-temperature waterlogging by simulating different flooding depths and water temperatures. It provides data support for alfalfa cultivation under high-temperature waterlogging stress and theoretical guidance for reducing economic losses through timely harvesting during waterlogging. A two-factor randomized block design was adopted with three flooding levels (W1: 0 cm, level with soil surface; W2: 10 cm above soil surface; and W3: 20 cm above soil surface) and four flooding water temperatures (25, 30, 35, and 40 ℃), resulting in 12 treatment groups (3 levels × 4 temperatures). The control group (CK) was maintained at air temperature (25 ± 2) ℃ and 70% field capacity. Samples were collected on the 2nd, 4th, and 6th days to measure alfalfa nutritional quality, metabolic products, root system vitality, antioxidant enzyme activity, and anaerobic respiration enzyme activity. The results showed that: 1) Flooding water temperature, flooding level, and flooding time significantly affected alfalfa physiological and biochemical indicators. 2) Flood water temperature had a more significant impact on alfalfa physiological and biochemical indicators than flooding level, especially under high-temperature conditions (40 ℃) where alfalfa physiological functions were almost completely impaired. This indicated that high temperature is a key environmental factor leading to rapid alfalfa death during summer waterlogging. 3) With the increase in flooding depth, water temperature, and flooding time, the alfalfa survival time shortened; furthermore, crude protein (CP), ether extract (EE), relative feed value (RFV), soluble sugars in the roots (SSR), and root system vitality (RSV) significantly decreased, and malondialdehyde (MDA) content in the leaves significantly increased. Ascorbate peroxidase (APX) activities in the leaves and alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) in the roots initially increased, but then decreased. 4) Under mild stress conditions (e.g., 25 ℃ flood water temperature, W1 flooding level, and 2 days of flooding time), CP, EE, SSR, and RSV significantly increased compared to CK, while neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents significantly decreased. Specifically, CP, SSR, and RSV reached their highest levels in the 25 ℃ + W1 treatment group on the 2nd day, NDF and ADF reached their lowest levels in the 25 ℃ + W1 treatment group on the 4th and 2nd day, and RFV peaked in the 25 ℃ + W2 treatment group on the 2nd day. These findings indicate that there is a transient quality optimization window that provides a critical time node for disaster harvesting. Although previous studies have revealed the effects of temperature and flooding time on alfalfa under single stress conditions, the physiological and biochemical response mechanisms and quality changes under the combined stress of flood depth, water temperature, and time remain unclear. This study elucidates the physiological and biochemical response mechanisms and quality changes in alfalfa under the combined stress of flood depth, water temperature, and time. It also provides a theoretical framework and data support for breeding waterlogging-resistant alfalfa varieties, decision-making for disaster harvesting, and precision cultivation management under high-temperature waterlogged conditions.