One overlooked aspect of the cardiovascular system’s reaction to acid-base disturbances is how lymphatic vessels respond to a metabolic challenge. Blood vessels are well known to vasodilate in acidotic conditions through various cellular mechanisms. Whether extracellular pH similarly influences lymphatic vessels is particularly relevant given that the lymph they transport can originate from tumour environments and areas of infection and inflammation where interstitial pCO2, [H+], and [HCO3-] can significantly deviate from systemic levels. We aimed to establish whether extracellular acidification reduces the contractile activity of human collecting lymphatic vessels. Human lymphatic vessels sourced from surgical patients (obtained after informed consent from each patient prior to surgery) were investigated. The use of human collecting vessels was approved by the ethical committee for the Danish Regional Health Authority and the study was conducted in accordance with the standards set by the Helsinki Declaration. Segments of thoracic duct (diameter) or intestinal lymphatics (diameter) were mounted in wire or pressure myographs for isometric force or isobaric diameter recording. The extracellular pH of the physiological salt solution immersing the vessels was maintained at different levels by altering the composition of CO2 and HCO3- in the solutions; control levels (pH 7.4, 22 mM HCO3-, 5% CO2) were compared with varying grades of metabolic acidosis (pH 7.1 and 6.8 obtained by lowering [HCO3-]) and respiratory acidosis (pH 7.1 obtained by increasing pCO2). Spontaneous and agonist-induced contractile behaviour (in response to noradrenaline and serotonin) were investigated in parallel under all four conditions. Paired comparison between pH 7.4 conditions and acidosis demonstrated that lowering extracellular pH inhibited phasic contractile activity of the thoracic duct whereas agonist-stimulated contractility was generally more robust. The isometrically-assessed contractility was attenuated when pH was lowered: at pH 6.8, all spontaneous phasic activity ceased, noradrenaline- and serotonin-stimulated tonic constriction was lowered by ≈50% and agonist-stimulated phasic activity was highly abrogated. Agonist-induced tonic contractile responses were minimally affected during moderate acidosis (metabolic and respiratory acidosis pH 7.1) whereas phasic contractile activity was substantially lowered. In the absence of agonists, pressurized thoracic duct segments displayed myogenic tone at transmural pressures of 7 and 20 mmHg. At both pressure levels, metabolic acidosis pH 6.8 significantly lowered myogenic tone (spontaneous and noradrenaline-stimulated) and phasic activity. We propose that phasic contractile activity of lymphatic vessel – which is considered the primary mechanisms for lymph propulsion – is attenuated by pathophysiologically realistic degrees of acidosis.