Objective: To examine the acute effects of isometric exercise of different intensities on pain perception in individuals with chronic lateral epicondylalgia. Materials and Methods: Participants performed 3 experimental tasks completed in a randomized order on separate days: control (no exercise) and isometric wrist extension (10×15 s) at load 20% below (infrathreshold), and 20% above (suprathreshold) an individual’s pain threshold. Self-reported pain intensity (11-point numeric rating scales), pressure pain threshold, and pain-free grip were assessed by a blinded examiner before, immediately after, and 30 minutes after task performance. Relation analysis between pain ratings and clinical variables, including pain and disability and kinesiophobia was performed. Results: Twenty-four individuals with unilateral lateral epicondylalgia of median 3-month duration participated. Pain intensity during contraction was significantly higher during suprathreshold exercise than infrathreshold exercise (mean difference in numeric rating scale 1.0; 95% confidence interval, 0.4-1.5; P=0.002). Pain intensity during suprathreshold exercise was significantly correlated with pain and disability (R=0.435, P=0.034) and kinesiophobia (R=0.556, P=0.005). Pain intensity was significantly higher immediately after performance of suprathreshold exercise, compared with infrathreshold exercise (P=0.01) and control (P<0.001) conditions, whereas infrathreshold exercise and control conditions were comparable. Thirty minutes later, pain levels remained significantly higher for suprathreshold exercise compared with infrathreshold exercise (P=0.043). Pressure pain threshold and pain-free grip showed no significant effects of time, condition, or time×condition (P>0.05). Discussion: Individuals with lateral epicondylalgia demonstrated increased pain intensity after an acute bout of isometric exercise performed at an intensity above, but not below, their individual pain threshold. Further investigation is needed to determine whether measurement of an individual’s exercise induced pain threshold may be important in reducing symptom flares associated with exercise.