Blood lactate measurements are used widely in horses to assess athletic performance, but there are few published data on using lactate as a guide for endurance improvement. The velocity at which blood lactate concentration ([LA]) of 4 mmol/I is reached (v4) is widely used to determine fitness. In an earlier study of v4, exercise at low intensities for longer duration was more effective at improving endurance than shorter, higher intensities. However, the prescription was unchanged during the study (6 weeks). We hypothesised that, to produce greater improvement in v4 in the same time frame, it is necessary to adjust conditioning regimes more frequently. Six horses underwent an initial treadmill-based standard exercise test (SET). A regression analysis [LA]-speed relationship was used to calculate v2. Horses were then conditioned for 45 min at their calculated v2 3 times/week (6% incline). Every 2 weeks the horses performed an SET to evaluate the v2 conditioning protocol which ended with SET 4. Mean v4 increased 17% from SET 1 to SET 4 (P = 0.003; 5.8 ± 0.3 to 6.8 ± 0.4 m/s). This study demonstrates that a lactate-guided conditioning programme can significantly enhance endurance performance over a 6-week time peroid when the conditioning protocol is adjusted every 2 weeks based on v4 improvement.