While prolonged standing has shown to be detrimentally associated with musculoskeletal symptoms, exposure limits and underlying mechanisms are not well understood. We systematically reviewed evidence from laboratory studies on musculoskeletal symptom development during prolonged (≥20 min) uninterrupted standing, quantified acute dose-response associations and described underlying mechanisms. Peer-reviewed articles were systematically searched for. Data from included articles were tabulated, and dose-response associations were statistically pooled. A linear interpolation of pooled dose-response associations was performed to estimate the duration of prolonged standing associated with musculoskeletal symptoms with a clinically relevant intensity of ≥9 (out of 100). We included 26 articles (from 25 studies with 591 participants), of which the majority examined associations of prolonged standing with low back and lower extremity symptoms. Evidence on other (e.g., upper limb) symptoms was limited and inconsistent. Pooled dose-response associations showed that clinically relevant levels of low back symptoms were reached after 71 min of prolonged standing, with this shortened to 42 min in those considered pain developers. Regarding standing-related low back symptoms, consistent evidence was found for postural mechanisms (i.e., trunk flexion and lumbar curvature), but not for mechanisms of muscle fatigue and/or variation in movement. Blood pooling was the most consistently reported mechanism for standing-related lower extremity symptoms. Evidence suggests a detrimental association of prolonged standing with low back and lower extremity symptoms. To avoid musculoskeletal symptoms (without having a-priori knowledge on whether someone will develop symptoms or not), dose-response evidence from this study suggests a recommendation to refrain from standing for prolonged periods >40 min. Interventions should also focus on underlying pain mechanisms.